Chemical compositions and uses thereof

ABSTRACT

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell that are adaptable for use with existing sequencing technologies.

BACKGROUND OF THE INVENTION

This application is a continuation of U.S. patent application Ser. No.16/272,487, filed Feb. 11, 2019. U.S. patent application Ser. No.16/272,487 claims priority to, and the benefit of, U.S. ProvisionalApplication No. 62/629,180, filed Feb. 12, 2018 and U.S. ProvisionalApplication No. 62/771,212, filed Nov. 26, 2018. The contents of each ofthe aforementioned patent applications are incorporated herein byreference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 15, 2021, isnamed “NATE-037_C02US_SeqList.txt” and is about 50,019 bytes in size.

BACKGROUND OF THE INVENTION

Standard immunohistochemical and in situ hybridization methods allow forsimultaneous detection of, at most, six to ten protein or nucleic acidtargets, with three to four targets being typical. There exists a needfor probes, compositions, methods, and kits for simultaneous,multiplexed detection and quantification of protein and/or nucleic acidexpression in a user-defined region of a tissue, user-defined cell,and/or user-defined subcellular structure within a cell. Furthermore,there is a need for such systems to be adaptable for use with existingsequencing technologies that are already available to a large number ofend users.

SUMMARY OF THE INVENTION

The present disclosure relates to probes, compositions, methods, andkits for simultaneous, multiplexed, spatial detection and quantificationof protein and/or nucleic acid expression in a user-defined region of atissue, user-defined cell, and/or user-defined subcellular structurewithin a cell.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) ligating to the released identifieroligonucleotide at least one nucleic acid adapter, wherein the nucleicacid adapter comprises a nucleic acid sequence which identifies thespecific location of the tissue sample from which the identifieroligonucleotide was released, a nucleic acid sequence comprising aunique molecular identifier, a first amplification primer binding site,a second amplification primer binding site and optionally, a constantnucleic acid sequence to minimize ligation bias; (5) amplifying theligation product produced in step (4); and (6) Identifying the releasedidentifier oligonucleotide by sequencing the amplified products producedin step (5), thereby spatially detecting the at least one target analytein the at least one cell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) ligating to the released identifieroligonucleotide at least one nucleic acid adapter, wherein the nucleicacid adapter comprises: a nucleic acid sequence comprising a uniquemolecular identifier, a first amplification primer binding site, asecond amplification primer binding site and optionally, a constantnucleic acid sequence to minimize ligation bias, and wherein at leastone of the first or second amplification primer binding sites identifiesthe specific location of the tissue sample from which the identifieroligonucleotide was released; (5) amplifying the ligation productproduced in step (4); and (6) Identifying the released identifieroligonucleotide by sequencing the amplified products produced in step(5), thereby spatially detecting the at least one target analyte in theat least one cell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) ligating to the released identifieroligonucleotide at least one nucleic acid adapter, wherein the nucleicacid adapter comprises: a nucleic acid sequence comprising a uniquemolecular identifier, a first amplification primer binding site, asecond amplification primer binding site and optionally, a constantnucleic acid sequence to minimize ligation bias; (5) amplifying theextension product produced in step (4) using a first amplificationprimer capable of binding to the first amplification primer binding siteand a second amplification primer capable of binding to the secondamplification primer binding site, wherein at least one of theamplification primers comprise a nucleic acid sequence which identifiesthe specific location of the tissue sample from which the identifieroligonucleotide was released; and (6) Identifying the releasedidentifier oligonucleotide by sequencing the amplified products producedin step (5), thereby spatially detecting the at least one target analytein the at least one cell in a tissue sample.

The nucleic acid adapter of step (4) can be a partially double-strandednucleic acid molecule. A partially double-stranded nucleic acid adaptercan comprise a double-stranded annealed region, a first single-strandedmismatched region and a second single-stranded mismatched region. Thefirst single-stranded mismatched region and the second single strandedmismatched region can be present on opposing sides of thedouble-stranded annealed region.

The nucleic acid sequence which identifies the specific location of thetissue sample from which the identifier oligonucleotide was released canbe present in the double-stranded annealed region of a partiallydouble-stranded nucleic acid adapter.

The constant nucleic acid sequence to minimize ligation bias can bepresent in the double-stranded annealed region of a partiallydouble-stranded nucleic acid adapter.

A unique molecular identifier can be present in at least one of thefirst or second single-stranded mismatched regions of a partiallydouble-stranded nucleic acid adapter.

The first amplification primer binding site can be present in the firstsingle-stranded mismatched region of a partially double-stranded nucleicacid adapter and the second amplification primer binding site can bepresent in the second single-stranded mismatched region of the samepartially double-stranded nucleic acid adapter.

The methods of the present disclosure described in the preceding canfurther comprise prior to step (4), performing an end repair reaction.The methods can also further comprise prior to step (4), performing atailing reaction to attach a single nucleotide overhang to the 3′ endsof the identifier oligonucleotide. The methods can further comprise,prior to step (4), performing an end repair reaction and a tailingreaction to attach a single nucleotide overhang to the 3′ ends of theidentifier oligonucleotide. The tailing reaction and the end repairreaction can be performed sequentially or concurrently.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a first amplification primer binding site, anda unique nucleic acid sequence which identifies the target analyte boundto the target binding domain; (2) providing a force to a location of thetissue sample sufficient to release the identifier oligonucleotide; (3)collecting the released identifier oligonucleotide; (4) ligating to thereleased identifier oligonucleotide at least one nucleic acid adapter,wherein the nucleic acid adapter comprises a nucleic acid sequence whichidentifies the specific location of the tissue sample from which theidentifier oligonucleotide was released, a nucleic acid sequencecomprising a unique molecular identifier, a second amplification primerbinding site and optionally, a constant nucleic acid sequence tominimize ligation bias; (5) amplifying the ligation product produced instep (4); and (6) Identifying the released identifier oligonucleotide bysequencing the amplified products produced in step (5), therebyspatially detecting the at least one target analyte in the at least onecell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises: a first amplification primer binding site anda unique nucleic acid sequence which identifies the target analyte boundto the target binding domain; (2) providing a force to a location of thetissue sample sufficient to release the identifier oligonucleotide; (3)collecting the released identifier oligonucleotide; (4) ligating to thereleased identifier oligonucleotide at least one nucleic acid adapter,wherein the nucleic acid adapter comprises a nucleic acid sequencecomprising a unique molecular identifier, a second amplification primerbinding site, and optionally, a constant nucleic acid sequence tominimize ligation bias; (5) amplifying the extension product produced instep (4) using a first amplification primer capable of binding to thefirst amplification primer binding site and a second amplificationprimer capable of binding to the second amplification primer bindingsite, wherein at least one of the amplification primers comprise anucleic acid sequence which identifies the specific location of thetissue sample from which the identifier oligonucleotide was released;and (6) Identifying the released identifier oligonucleotide bysequencing the amplified products produced in step (5), therebyspatially detecting the at least one target analyte in the at least onecell in a tissue sample.

The nucleic acid adapter of step (4) can be a partially double-strandednucleic acid molecule. A partially double-stranded nucleic acid adaptercan comprise a double-stranded annealed region and a single-strandedmismatched region.

The nucleic acid sequence which identifies the specific location of thetissue sample from which the identifier oligonucleotide was released canbe present in the double-stranded annealed region of a partiallydouble-stranded nucleic acid adapter.

The constant nucleic acid sequence to minimize ligation bias can bepresent in the double-stranded annealed region of a partiallydouble-stranded nucleic acid adapter. The constant nucleic acid sequencecan also comprise a cleavable moiety. The cleavable moiety can be anenzymatically cleavable moiety. The enzymatically cleavable moiety canbe a USER sequence.

A unique molecular identifier can be present in the single-strandedmismatched region of a partially double-stranded nucleic acid adapter.

The second amplification primer binding site can be present in thesingle-stranded mismatched region of a partially double-stranded nucleicacid adapter.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a first amplification primer binding site, anda unique nucleic acid sequence which identifies the target analyte boundto the target binding domain; (2) providing a force to a location of thetissue sample sufficient to release the identifier oligonucleotide; (3)collecting the released identifier oligonucleotide; (4) hybridizing tothe released identifier oligonucleotide a single stranded nucleic acidtemplate, wherein the nucleic acid template comprises a regioncomplementary to the unique nucleic acid sequence of the identifieroligonucleotide, a nucleic acid sequence comprising a unique molecularidentifier, a nucleic acid sequence complementary to a secondamplification primer binding site and optionally, an affinity molecule;(5) extending the identifier oligonucleotide of step (4) to form anextension product complementary to the single stranded nucleic acidtemplate, wherein the extension product comprises the identifieroligonucleotide, the nucleic acid sequence complementary to the uniquemolecular identifier, and the second amplification primer binding site;(6) amplifying the extension product produced in step (5) using a firstamplification primer capable of binding to the first amplificationprimer binding site and a second amplification primer capable of bindingto the second amplification primer binding site, wherein at least one ofthe amplification primers comprise a nucleic acid sequence whichidentifies the specific location of the tissue sample from which theidentifier oligonucleotide was released; (7) Identifying the releasedidentifier oligonucleotide by sequencing the amplified products producedin step (6), thereby spatially detecting the at least one target analytein the at least one cell in a tissue sample.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a first amplification primer binding site anda unique nucleic acid sequence which identifies the target analyte boundto the target binding domain; (2) providing a force to a location of thetissue sample sufficient to release the identifier oligonucleotide; (3)collecting the released identifier oligonucleotide; (4) hybridizing tothe released identifier oligonucleotide a single stranded nucleic acidtemplate, wherein the nucleic acid template comprises a regioncomplementary to the unique nucleic acid sequence of the identifieroligonucleotide, a nucleic acid sequence which identifies the specificlocation of the tissue sample from which the identifier oligonucleotidewas released, a nucleic acid sequence comprising a unique molecularidentifier, a nucleic acid sequence complementary to a secondamplification primer binding site and optionally, an affinity molecule;(5) extending the identifier oligonucleotide of step (4) to form anextension product complementary to the single stranded nucleic acidtemplate, wherein the extension product comprises the identifieroligonucleotide, the nucleic acid sequence complementary to the nucleicacid sequence which identifies the specific location of the tissuesample from which the identifier oligonucleotide was released, thenucleic acid sequence complementary to the unique molecular identifierand the second amplification primer binding site; (6) amplifying theextension product produced in step (5); and (7) Identifying the releasedidentifier oligonucleotide by sequencing the amplified products producedin step (6), thereby spatially detecting the at least one target analytein the at least one cell in a tissue sample.

The single stranded nucleic acid template can further comprise anaffinity molecule. In aspects in which the single stranded nucleic acidtemplate comprises an affinity molecule, the methods of the presentdisclosure described in the preceding can further comprise an affinitypurification step between steps (4) and (5).

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide, a nucleicacid sequence which identifies the specific location of the tissuesample from which the identifier oligonucleotide was released and afirst amplification primer binding site, and wherein the second nucleicacid probe comprises a nucleic acid complementary to a portion of theidentifier oligonucleotide, a nucleic acid sequence comprising a uniquemolecular identifier and a second amplification primer binding site, andwherein the first the second nucleic acid probes hybridize to theidentifier oligonucleotide such that the first and the second nucleicacid probes are adjacent but not overlapping; (5) performing nick repairsuch that the hybridized first and second nucleic acid probes areligated together; (6) amplifying the ligation product produced in step(5); and (7) Identifying the released identifier oligonucleotide bysequencing the amplified products produced in step (6), therebyspatially detecting the at least one target analyte in the at least onecell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide and a firstamplification primer binding site, and wherein the second nucleic acidprobe comprises a nucleic acid complementary to a portion of theidentifier oligonucleotide and a second amplification primer bindingsite, and wherein at least one of the first or second nucleic acidprobes comprises a nucleic acid sequence comprising a unique molecularidentifier, and wherein at least one of the first or second nucleic acidprobes comprises a nucleic acid sequence which identifies the specificlocation of the tissue sample from which the identifier oligonucleotidewas released, and wherein the first and the second nucleic acid probeshybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are adjacent but not overlapping; (5)performing nick repair such that the hybridized first and second nucleicacid probes are ligated together; (6) amplifying the ligation productproduced in step (5); and (7) Identifying the released identifieroligonucleotide by sequencing the amplified products produced in step(6), thereby spatially detecting the at least one target analyte in theat least one cell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide, a nucleicacid sequence which identifies the specific location of the tissuesample from which the identifier oligonucleotide was released and afirst amplification primer binding site, and wherein the second nucleicacid probe comprises a nucleic acid complementary to a portion of theidentifier oligonucleotide, a nucleic acid sequence comprising a uniquemolecular identifier and a second amplification primer binding site, andwherein the first and the second nucleic acid probes hybridize to theidentifier oligonucleotide such that the first and the second nucleicacid probes are not adjacent and are not overlapping; (5) performing agap extension and nick repair reaction such that the hybridized firstand second nucleic acid probes are ligated together; (6) amplifying theligation product produced in step (5); and (7) Identifying the releasedidentifier oligonucleotide by sequencing the amplified products producedin step (6), thereby spatially detecting the at least one target analytein the at least one cell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide and a firstamplification primer binding site, and wherein the second nucleic acidprobe comprises a nucleic acid complementary to a portion of theidentifier oligonucleotide and a second amplification primer bindingsite, and wherein at least one of the first or second nucleic acidprobes comprises a nucleic acid sequence comprising a unique molecularidentifier, and wherein at least one of the first or second nucleic acidprobes comprises a nucleic acid sequence which identifies the specificlocation of the tissue sample from which the identifier oligonucleotidewas released, and wherein the first and the second nucleic acid probeshybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are not adjacent and are not overlapping; (5)performing a gap extension and nick repair reaction such that thehybridized first and second nucleic acid probes are ligated together;(6) amplifying the ligation product produced in step (5); and (7)Identifying the released identifier oligonucleotide by sequencing theamplified products produced in step (6), thereby spatially detecting theat least one target analyte in the at least one cell in a tissue sample.

In aspects in which the nucleic acid sequence which identifies thespecific location of the tissue sample from which the identifieroligonucleotide was released is located on the first nucleic acid probe,the nucleic acid sequence which identifies the specific location of thetissue sample from which the identifier oligonucleotide was released canbe located 5′ to the first amplification primer binding site.

In aspects in which a unique molecular identifier is located on thesecond nucleic acid probe, the unique molecular identifier can belocated 3′ to the second amplification primer binding site.

In aspects in which the nucleic acid sequence which identifies thespecific location of the tissue sample from which the identifieroligonucleotide was released and a unique molecular identifier arepresent in the first nucleic acid probe, the nucleic acid sequence whichidentifies the specific location of the tissue sample from which theidentifier oligonucleotide was released and a unique molecularidentifier can be located 5′ to the first amplification primer bindingsite.

In aspects in which the nucleic acid sequence which identifies thespecific location of the tissue sample from which the identifieroligonucleotide was released and a unique molecular identifier arepresent in the second nucleic acid probe, the nucleic acid sequencewhich identifies the specific location of the tissue sample from whichthe identifier oligonucleotide was released and the unique molecularidentifier can be located 3′ to the second amplification primer bindingsite.

In aspects in which a unique molecular identifier is present in thefirst nucleic acid probe and the nucleic acid sequence which identifiesthe specific location of the tissue sample from which the identifieroligonucleotide was released is present in the second nucleic acidprobe, the unique molecular identifier can be located 5′ to the firstamplification primer binding site and the nucleic acid sequence whichidentifies the specific location of the tissue sample from which theidentifier oligonucleotide was released can be located 3′ to the secondamplification binding site.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide, a nucleicacid sequence which identifies the specific location of the tissuesample from which the identifier oligonucleotide was released, a firstamplification primer binding site, a nucleic acid sequence comprising afirst unique molecular identifier and a first flow cell binding site,and wherein the second nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide, a nucleicacid sequence comprising a second unique molecular identifier, a nucleicacid sequence comprising a third unique molecular identifier and asecond flow cell binding site, and wherein the first and the secondnucleic acid probes hybridize to the identifier oligonucleotide suchthat the first and the second nucleic acid probes are adjacent but notoverlapping; (5) performing nick repair such that the hybridized firstand second nucleic acid probes are ligated together; (6) amplifying theligation product produced in step (5); and (7) Identifying the releasedidentifier oligonucleotide by sequencing the amplified products producedin step (6), thereby spatially detecting the at least one target analytein the at least one cell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide, a firstamplification primer binding site, a nucleic acid sequence comprising afirst unique molecular identifier and a first flow cell binding site,and wherein the second nucleic acid probe comprises a nucleic acidcomplementary to a portion of the identifier oligonucleotide, a nucleicacid sequence comprising a second unique molecular identifier and asecond flow cell binding site, and wherein at least one of the first orsecond nucleic acid probes comprises a nucleic acid sequence comprisinga third unique molecular identifier, and wherein at least one of thefirst or second nucleic acid probes comprises a nucleic acid sequencewhich identifies the specific location of the tissue sample from whichthe identifier oligonucleotide was released, and wherein the first andthe second nucleic acid probes hybridize to the identifieroligonucleotide such that the first and the second nucleic acid probesare adjacent but not overlapping; (5) performing nick repair such thatthe hybridized first and second nucleic acid probes are ligatedtogether; (6) amplifying the ligation product produced in step (5); and(7) Identifying the released identifier oligonucleotide by sequencingthe amplified products produced in step (6), thereby spatially detectingthe at least one target analyte in the at least one cell in a tissuesample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to the identifier oligonucleotide, a nucleic acid sequencewhich identifies the specific location of the tissue sample from whichthe identifier oligonucleotide was released, a first amplificationprimer binding site, a nucleic acid sequence comprising a first uniquemolecular identifier and a first flow cell binding site, and wherein thesecond nucleic acid probe comprises a nucleic acid complementary to theidentifier oligonucleotide, a nucleic acid sequence comprising a secondunique molecular identifier, a nucleic acid sequence comprising a thirdunique molecular identifier and a second flow cell binding site, andwherein the first and the second nucleic acid probes hybridize to theidentifier oligonucleotide such that the first and the second nucleicacid probes are not adjacent and are not overlapping; (5) performing agap extension and nick repair reaction such that the hybridized firstand second nucleic acid probes are ligated together; (6) amplifying theligation product produced in step (5); and (7) Identifying the releasedidentifier oligonucleotide by sequencing the amplified products producedin step (6), thereby spatially detecting the at least one target analytein the at least one cell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises a nucleic acidcomplementary to the identifier oligonucleotide, a first amplificationprimer binding site, a nucleic acid sequence comprising a first uniquemolecular identifier and a first flow cell binding site, and wherein thesecond nucleic acid probe comprises a nucleic acid complementary to theidentifier oligonucleotide, a nucleic acid sequence comprising a secondunique molecular identifier and a second flow cell binding site, andwherein at least one of the first or second nucleic acid probescomprises a nucleic acid sequence comprising a third unique molecularidentifier, and wherein at least one of the first or second nucleic acidprobes comprises a nucleic acid sequence which identifies the specificlocation of the tissue sample from which the identifier oligonucleotidewas released, and wherein the first and the second nucleic acid probeshybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are not adjacent and are not overlapping; (5)performing a gap extension and nick repair reaction such that thehybridized first and second nucleic acid probes are ligated together;(6) amplifying the ligation product produced in step (5); and (7)Identifying the released identifier oligonucleotide by sequencing theamplified products produced in step (6), thereby spatially detecting theat least one target analyte in the at least one cell in a tissue sample.

In aspects in which the nucleic acid sequence which identifies thespecific location of the tissue sample from which the identifieroligonucleotide was released and the first unique molecular identifierare present in the first nucleic acid probe, the nucleic acid sequencewhich identifies the specific location of the tissue sample from whichthe identifier oligonucleotide was released and the first uniquemolecular identifier can be located 5′ to the first flow cell bindingsite.

In aspects in which the second and the third unique molecularidentifiers are present in the second nucleic acid probe, the second andthe third unique molecular identifiers can be located 3′ to the secondflow cell binding site.

In some aspects, the first unique molecular identifier can be present inthe first nucleic acid probe and can be located 5′ to the first flowcell binding site. In other aspects, the second unique molecularidentifier can be present in the second nucleic acid probe and can belocated 3′ to the second flow cell binding site.

In some aspects, the nucleic acid sequence which identifies the specificlocation of the tissue sample from which the identifier oligonucleotidewas released and the third unique molecular identifier can be present inthe first nucleic acid probe and can be located 5′ to the first flowcell binding site.

In some aspects, the nucleic acid sequence which identifies the specificlocation of the tissue sample from which the identifier oligonucleotidewas released and the third unique molecular identifier can be present inthe second nucleic acid probe and can be located 3′ to the second flowcell binding site.

In some aspect, the third unique molecular identifier can be present inthe first nucleic acid probe and can be located 5′ to the first flowcell binding site. In this same aspect, the nucleic acid sequence whichidentifies the specific location of the tissue sample from which theidentifier oligonucleotide was released can be present in the secondnucleic acid probe and can be located 3′ to the second flow cell bindingsite.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain, anucleic acid sequence comprising a unique molecular identifier, a firstamplification primer binding site, and a second amplification primerbinding site; (2) providing a force to a location of the tissue samplesufficient to release the identifier oligonucleotide; (3) collecting thereleased identifier oligonucleotide; (4) amplifying the releasedidentifier oligonucleotide using a first amplification primer capable ofbinding to the first amplification primer binding site and a secondamplification primer capable of binding to the second amplificationprimer binding site, wherein at least one of the amplification primerscomprises a nucleic acid sequence which identifies the specific locationof the tissue sample from which the identifier oligonucleotide wasreleased; (5) Identifying the released identifier oligonucleotide bysequencing the amplified products produced in step (4), therebyspatially detecting the at least one target analyte in the at least onecell in a tissue sample.

The present disclosure also provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain, afirst amplification primer binding site and a second amplificationprimer binding site; (2) providing a force to a location of the tissuesample sufficient to release the identifier oligonucleotide; (3)collecting the released identifier oligonucleotide; (4) amplifying thereleased identifier oligonucleotide using a first amplification primercapable of binding to the first amplification primer binding site and asecond amplification primer capable of binding to the secondamplification primer binding site, wherein at least one of theamplification primers comprises a nucleic acid sequence which identifiesthe specific location of the tissue sample from which the identifieroligonucleotide was released, and wherein at least one of theamplification primers comprises a nucleic acid sequence comprising aunique molecular identifier; and (5) Identifying the released identifieroligonucleotide by sequencing the amplified products produced in step(4), thereby spatially detecting the at least one target analyte in theat least one cell in a tissue sample.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain and acapture probe binding site; (2) providing a force to a location of thetissue sample sufficient to release the identifier oligonucleotide; (3)collecting the released identifier oligonucleotide; (4) hybridizing tothe released identifier oligonucleotide a capture probe, wherein thecapture probe comprises an affinity molecule and a region complementaryto the capture probe binding site; and (5) Identifying the releasedidentifier oligonucleotide by sequencing the amplified hybridizedproduct produced in step (4), thereby spatially detecting the at leastone target analyte in the at least one cell in a tissue sample.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain, acapture probe binding site and a multiplexing probe binding site; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a capture probe and a multiplexing probe, wherein thecapture probe comprises an affinity molecule and a region complementaryto the capture probe binding site, and wherein the multiplexing probecomprises a nucleic acid sequence which identifies the specific locationof the tissue sample from which the identifier oligonucleotide wasreleased and a region complementary to the multiplexing probe bindingsite; and (5) Identifying the released identifier oligonucleotide bysequencing the hybridized product produced in step (4), therebyspatially detecting the at least one target analyte in the at least onecell in a tissue sample.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain; (2)providing a force to a location of the tissue sample sufficient torelease the identifier oligonucleotide; (3) collecting the releasedidentifier oligonucleotide; (4) hybridizing to the released identifieroligonucleotide a first nucleic acid probe and a second nucleic acidprobe, wherein the first nucleic acid probe comprises: a nucleic acidcomplementary to a portion of the identifier oligonucleotide, a nucleicacid sequence comprising a unique molecular identifier, a firstamplification primer binding site, and wherein the second nucleic acidprobe comprises: a nucleic acid complementary to a portion of theidentifier oligonucleotide, and a second amplification primer bindingsite, and wherein the first and the second nucleic acid probes hybridizeto the identifier oligonucleotide such that the first and the secondnucleic acid probes are adjacent but not overlapping; (5) ligating thehybridized first and second nucleic acid probes together; (6) amplifyingthe ligation product produced in step (5); and (7) identifying thereleased identifier oligonucleotide by sequencing the amplified productsproduced in step (6), thereby spatially detecting the at least onetarget analyte in the at least one cell in a tissue sample.

The present disclosure provides a method for spatially detecting atleast one target analyte in at least one cell from a tissue samplecomprising: (1) contacting at least one target analyte in at least onecell in a tissue sample with at least one probe comprising a targetbinding domain and an identifier oligonucleotide, wherein the identifieroligonucleotide comprises: a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain, anucleic acid sequence comprising a unique molecular identifier, a firstamplification primer binding site, and a second amplification primerbinding site; (2) providing a force to a location of the tissue samplesufficient to release the identifier oligonucleotide; (3) collecting thereleased identifier oligonucleotide; (4) amplifying the collectedidentifier oligonucleotide; (5) Identifying the released identifieroligonucleotide by sequencing the amplified products produced in step(4), thereby spatially detecting the at least one target analyte in theat least one cell in a tissue sample.

A In all methods of the present disclosure, the ligation process can bea nick ligation process. The nick ligation process can be a nick repairprocess.

In all methods of the present disclosure, the sequencing can be anenzyme free sequencing method.

In all methods of the present disclosure, the identifier oligonucleotidecan be double-stranded. In aspects in which the identifieroligonucleotide is double-stranded, at least one of the two strands ofthe identifier oligonucleotide can comprise at least two separatenucleic acid molecules. Alternatively, at least one 3′ end of anidentifier oligonucleotide can comprise a single nucleotide overhang.

In all methods of the present disclosure, the identifier oligonucleotidecan be single-stranded.

In all methods of the present disclosure, the unique nucleic acidsequence which identifies the target analyte bound to a target bindingdomain can comprise between about 5 nucleotides and about 40 nucleotidespreferably about 35 nucleotides, preferably still about 10 nucleotides.

In all methods of the present disclosure, the nucleic acid sequencewhich identifies the specific location of the tissue sample from whichthe identifier oligonucleotide was released can comprise between about 6nucleotides and about 15 nucleotides, preferably about 12 nucleotides,preferably still about 10 nucleotides.

In all methods of the present disclosure, at least one of a firstnucleic acid probe or a second nucleic acid probe can comprise anaffinity molecule. For example, at least one of a first nucleic acidprobe or a second nucleic acid probe can comprise a biotin.

In all methods of the present disclosure, an amplification primerbinding site can comprise between about 18 nucleotides and about 40nucleotides, preferably about 32 nucleotides, preferably still about 25nucleotides. An amplification primer binding site can comprise an i7sequence, wherein the i7 sequence comprises the sequence set forth inSEQ ID NO: 1. An amplification primer binding site can comprise an i5sequence, wherein the i5 sequence comprises the sequence set forth inSEQ ID NO: 2.

In all methods of the present disclosure, an amplification primer cancomprise a flow cell adapter sequence, wherein the flow cell adaptersequence is suitable for sequencing. An amplification primer cancomprise a P5 flow cell adapter sequence, wherein the P5 flow celladapter sequence comprises the sequence set forth in SEQ ID NO: 3. Anamplification primer can comprise a P7 flow cell adapter sequence,wherein the P7 flow cell adapter sequence comprises the sequence setforth in SEQ ID NO: 4.

In all methods of the present disclosure, a flow cell binding site cancomprise a flow cell adapter sequence, wherein the flow cell adaptersequence is suitable for sequencing. A flow cell binding site cancomprise a P5 flow cell adapter sequence, wherein the P5 flow celladapter sequence comprises the sequence set forth in SEQ ID NO: 3. Aflow cell binding site can comprise a P7 flow cell adapter sequence,wherein the P7 flow cell adapter sequence comprises the sequence setforth in SEQ ID NO: 4.

In all methods of the present invention, at least one of theamplification primers can comprise an affinity molecule. For example, atleast one of the amplification primers cam comprise a biotin.

In all methods of the present disclosure, amplification can compriseperforming PCR. Performing PCR can comprise an amplification primer.

An amplification primer can comprise a flow cell binding site. Anamplification primer can comprise a nucleic sequence which identifiesthe specific location of the tissue sample from which an identifieroligonucleotide was released. An amplification primer can comprise anucleic acid sequence complementary to an amplification primer bindingsite.

Any of the above aspects can be combined with any other aspect.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In the Specification, thesingular forms also include the plural unless the context clearlydictates otherwise; as examples, the terms “a,” “an,” and “the” areunderstood to be singular or plural and the term “or” is understood tobe inclusive. By way of example, “an element” means one or more element.Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromthe context, all numerical values provided herein are modified by theterm “about.”

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. The references cited hereinare not admitted to be prior art to the claimed invention. In the caseof conflict, the present Specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting. Other featuresand advantages of the disclosure will be apparent from the followingdetailed description and claim.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings.

FIG. 1 is a schematic of a two-ended adapter ligation method of thepresent disclosure.

FIG. 2 is a schematic of a one-ended adapter ligation method of thepresent disclosure.

FIG. 3 is a schematic of a templated-primer extension method of thepresent disclosure.

FIG. 4 is a schematic of a template-extended identifier oligonucleotideof the present disclosure.

FIG. 5 is a schematic of a short probe hybridization method of thepresent disclosure.

FIG. 6 is a schematic of a short probe hybridization method of thepresent disclosure.

FIG. 7 is a schematic of a short probe hybridization method of thepresent disclosure.

FIG. 8 is a schematic of a long probe hybridization method of thepresent disclosure.

FIG. 9 is a schematic of a long probe hybridization method of thepresent disclosure.

FIG. 10 is a schematic of a long probe hybridization method of thepresent disclosure.

FIG. 11 is a schematic of a direct-PCR method of the present disclosure.

FIG. 12 is a schematic of an enzyme free method of the presentdisclosure.

FIG. 13 is a schematic of a multiplexed enzyme free method of thepresent disclosure.

FIG. 14 is a schematic of a probe of the present disclosure indirectlybinding to a target nucleic acid.

FIG. 15 is a schematic of an identifier oligonucleotide-short nucleicacid probe complex of the present disclosure.

FIG. 16 is a schematic of a short probe hybridization method of thepresent disclosure.

FIG. 17 is a schematic of an identifier oligonucleotide-short nucleicacid probe complex of the present disclosure.

FIG. 18 is a schematic of a short probe hybridization method of thepresent disclosure.

FIG. 19 is a schematic of a direct-PCR method of the present disclosure.

FIG. 20 is a schematic overview of the methods of the presentdisclosure.

FIG. 21A, FIG. 21B, FIG. 21C and FIG. 21D show the spatial detection ofprotein target analytes using the methods of the present disclosure.

FIG. 22A, FIG. 22B, FIG. 22C and FIG. 22D show the spatial detection ofRNA target analytes using the methods of the present disclosure.

FIG. 23 shows the spatial detection of protein target analytes using themethods of the present disclosure.

FIG. 24 shows the spatial detection of RNA target analytes using themethods of the present disclosure.

FIG. 25 shows the spatial detection of protein target analytes using themethods of the present disclosure.

FIG. 26 is a schematic of a probe of the present disclosure. The nucleicacid sequence shown in FIG. 26 corresponds to SEQ ID NO: 175.

FIG. 27 shows the use of probe tiling in the methods of the presentdisclosure.

FIG. 28 shows the regions of interest selected on a tissue microarray.

FIG. 29 is a series of graphs showing the read depth achieved using themethods of the present disclosure.

FIG. 30 is a series of graphs showing the spatial detection of RNAtarget analytes in negative control samples using the methods of thepresent disclosure.

FIG. 31 is a series of graphs showing the spatial detection of RNAtarget analytes in a HEK293 sample (top panel) and a Jurkat cell sample(bottom panel) using the methods of the present disclosure.

FIG. 32 is a series of graphs showing the spatial detection of RNAtarget analytes in sixteen FFPE samples using the methods of the presentdisclosure.

FIG. 33 is a graph showing the spatial detection of RNA target analytesin a HEK293 sample using the methods of the present disclosure.

FIG. 34 is a graph showing the spatial detection of RNA target analytesin a Jurkat cell sample using the methods of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based in part on probes, compositions,methods, and kits for simultaneous, multiplexed spatial detection andquantification of protein and/or nucleic acid expression in auser-defined region of a tissue, user-defined cell, and/or user-definedsubcellular structure within a cell using existing sequencing methods.

The present disclosure provides a comparison of the identity andabundance of target proteins and/or target nucleic acids present in afirst region of interest (e.g., tissue type, a cell (including normaland abnormal cells), and a subcellular structure within a cell) and theidentity and abundance of target proteins and/or target nucleic acidspresent in a second region of interest. There is no pre-defined upperlimit to the number of regions of interest and comparisons that can bemade; the upper limit relates to the size of the region of interestrelative the size of the sample. As examples, when a single cellrepresents a region of interest, then a section may have hundreds tothousands of regions of interest; however, if a tissue section includesonly two cell types, then the section may have only two regions ofinterest (each including only one cell type).

The present disclosure provides a higher degree of multiplexing than ispossible with standard immunohistochemical or in situ hybridizationmethods. Standard immunohistochemical methods allow for maximalsimultaneous detection of six to ten protein targets, with three to fourprotein targets being more typical. Similarly, in situ hybridizationmethods are limited to simultaneous detection of fewer than ten nucleicacid targets. The present disclosure provides detection of largecombinations of nucleic acid targets and/or protein targets from adefined region of a sample. The present disclosure provides an increasein objective measurements by digital quantification and increasedreliability and consistency, thereby enabling comparison of resultsamong multiple centers.

Various compositions and methods of the present disclosure are describedin full detail herein.

In one aspect, the present disclosure provides compositions and methodsfor spatially detecting at least one target analyte in a sample usingthe probes of the present disclosure in a method herein referred to as a“two-ended adapter ligation method”.

A two ended-adapter ligation method of the present disclosure cancomprise: (1) contacting at least one target analyte in a sample with atleast one probe of the present disclosure. The probes and samples of thepresent disclosure are described in further detail herein. The at leastone target analyte can be a target protein or a target nucleic acid. Inthe aspect that the at least one target analyte is a target protein, theprobe can comprise a target binding domain that is a targetprotein-binding region that can specifically bind to a target protein ofinterest. In the aspect that the at least one target analyte is a targetnucleic acid, the probe can comprise a target nucleic acid-bindingregion that can directly or indirectly hybridize to a target nucleicacid of interest. The probe further comprises an identifieroligonucleotide. The identifier oligonucleotide can comprise a uniquenucleic acid sequence which identifies the target analyte bound to thetarget binding domain.

Following contacting the at least on target analyte with the at leastone probe, a two-ended adapter ligation method can further comprise: (2)providing a force to a location of the sample sufficient to release theidentifier oligonucleotide. In a non-limiting example, in aspects inwhich the probe comprises a photo-cleavable linker between theidentifier oligonucleotide and the target binding domain, aregion-of-interest is excited with light of a sufficient wavelengthcapable of cleaving the photo-cleavable linker.

Following release of the identifier oligonucleotide, a two-ended adapterligation method can further comprise: (3) collecting the releasedidentifier oligonucleotide. By directing the force only to a specificlocation in step (2), identifier oligonucleotides are only released fromprobes within that location and not from probes located outside thatlocation. Thus, identifier oligonucleotides are collected only forprobes that are bound to targets within that location, therebypermitting detection of the identities and quantities of the targets(proteins and/or nucleic acids) located only within that location.

Following collection of the released identifier oligonucleotide, atwo-ended adapter ligation method can further comprise: (4) ligating tothe released identifier oligonucleotide collected in step (3) at leastone nucleic acid adapter.

The nucleic acid adapter can comprise a nucleic acid sequence whichidentifies the specific location of the sample from which the identifieroligonucleotide was released. For example, if the identifieroligonucleotide was released from location of the sample designated “ROI#1”, the nucleic acid adapter would comprise a nucleic acid sequencethat corresponds to “ROI #1”.

The nucleic acid adapter can also comprise a unique molecularidentifier.

The nucleic acid adapter can also comprise a first amplification primerbinding site. In other aspects, the nucleic acid adapter can alsocomprise a second amplification primer binding site.

In some aspects, the nucleic acid adapter can also comprise a constantnucleic acid sequence to minimize ligation bias caused by differences insequences of particular identifier oligonucleotides.

The nucleic acid adapter can be a partially double-stranded nucleic acidmolecule. In aspects in which the nucleic acid adapter is partiallydouble-stranded, the nucleic acid adapter comprises a double-strandedannealed region, a first single-stranded mismatched region, and a secondsingle-stranded mismatched region. The first single-stranded mismatchedregion and the second single stranded mismatched region can be presenton opposing sides of the double-stranded annealed region.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a nucleic acid sequence which identifiesthe specific location of the sample from which the identifieroligonucleotide was released, the nucleic acid sequence which identifiesthe specific location of the sample from which the identifieroligonucleotide was released can be present in the double-strandedannealed region of the nucleic acid adapter.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a constant nucleic acid sequence tominimize ligation bias, the constant nucleic acid sequence to minimizeligation bias can be present in the double-stranded annealed region ofthe nucleic acid adapter.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a unique molecular identifier, the uniquemolecular identifier can be present in at least one of the first orsecond single-stranded mismatched regions of the nucleic acid adapter.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a first and a second amplification primerbinding site, the first amplification primer binding site can be presentin the first single-stranded mismatched region of the nucleic acidadapter and the second amplification primer binding site can be presentin the second single-stranded mismatched region of the nucleic acidadapter.

After ligation of the at least one nucleic acid adapter, a two-endedadapter ligation method can further comprise: (5) amplifying theligation product produced in step (4) using amplification primers thatbind to the first and second amplification primer binding sites; and (6)identifying the released oligonucleotide by sequencing the amplifiedproducts produced in step (5), thereby spatially detecting the at leastone target analyte in the sample.

A two-ended adapter ligation method of the present disclosure canfurther comprise, prior to step (4), performing an end repair reactionusing methods known in the art. The method can also further comprise,prior to step (4), performing a tailing reaction to attach a singlenucleotide overhang to the 3′ ends of the identifier oligonucleotideusing methods known in the art. In aspects, the end repair reaction andthe tailing reaction can be performed sequentially or concurrently.

In preferred aspects of a two-ended adapter ligation method, a nucleicacid adapter is ligated to both ends of the released and collectedidentifier oligonucleotide.

In other aspects of a two-ended adapter ligation method, at least one ofthe amplification primers used in step (5) to amplify the ligationproduct produced in step (4) comprises a nucleic acid sequence whichidentifies the specific location of the tissue sample form which theidentifier oligonucleotide was released. For example, if the identifieroligonucleotide was released from location of the sample designated “ROI#1”, at least one of the amplification primers would comprise a nucleicacid sequence that corresponds to “ROI #1”.

FIG. 1 shows a schematic of a preferred aspect of a two-ended adapterligation method of the present disclosure. In this aspect, the probecomprises a target binding domain comprising an antibody that binds to atarget protein. In the upper left panel, the probe binds to the targetprotein. In the upper right panel, a UV photo-cleavable linker locatedbetween the target binding domain and the identifier oligonucleotide iscleaved, releasing the identifier oligonucleotide. The identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target protein bound to the target binding domain. In thebottom panel, a nucleic acid adapter is ligated to both ends of theidentifier oligonucleotide. In this non-limiting example, the nucleicacid adapter is partially double-stranded and comprises adouble-stranded annealed region, a first single-stranded mismatchedregion, and a second single-stranded mismatched region. Present in thedouble-stranded annealed region is a constant nucleic acid sequence tominimize ligation bias and a nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released. Present in the first single-strandedmismatched region is a first amplification primer binding site. Presentin the second single-stranded mismatched region is a unique molecularidentifier and a second amplification primer binding site. Followingligation of the nucleic acid adapters to the identifier oligonucleotide,the product is amplified using amplification primers that bind the firstand the second amplification primer binding sites and sequenced toidentify the target protein bound by the probe.

In one aspect, the present disclosure provides a composition of anidentifier oligonucleotide dually ligated to two nucleic acid adaptersfor spatially detecting at least one target analyte in a sample. Anidentifier oligonucleotide dually ligated to two nucleic acid adapterscomprises an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence which iscapable of identifying a target analyte in a sample. Each end of theidentifier oligonucleotide is attached to a nucleic acid adaptermolecule, wherein the nucleic acid adapter molecule is partiallydouble-stranded and comprises a double-stranded annealed region, a firstsingle-stranded mismatched region and a second single-strandedmismatched region. The first single-stranded mismatched region and thesecond single stranded mismatched region are present on opposing sidesof the double-stranded annealed region. The double-stranded mismatchregion comprises a constant nucleic acid sequence to minimize ligationbias and a nucleic acid sequence nucleic acid sequence which is capableof identifying a specific location of a sample. The firstsingle-stranded mismatched region comprises a first amplification primerbinding site. The second single-stranded mismatched region comprises asecond amplification primer binding site and a nucleic acid sequencecomprising a unique molecular identifier. A schematic of an identifieroligonucleotide dually ligated to two nucleic acid adapters is shown inthe bottom panel of FIG. 1.

In another aspect, the present disclosure provides compositions andmethods for spatially detecting at least one target analyte in a sampleusing the probes of the present disclosure in a method herein referredto as a “one-ended adapter ligation method”.

A one-ended adapter ligation method of the present disclosure cancomprise: (1) contacting at least one target analyte in a sample with atleast one probe of the present disclosure. The at least one targetanalyte can be a target protein or a target nucleic acid. In the aspectthat the at least one target analyte is a target protein, the probe cancomprise a target binding domain that is a target protein-binding regionthat can specifically bind to a target protein of interest. In theaspect that the at least one target analyte is a target nucleic acid,the probe can comprise a target nucleic acid-binding region that candirectly or indirectly hybridize to a target nucleic acid of interest.The probe further comprises an identifier oligonucleotide. Theidentifier oligonucleotide can comprise a unique nucleic acid sequencewhich identifies the target analyte bound to the target binding domain.The identifier oligonucleotide can also comprise a first amplificationprimer binding site. In some aspects, the identifier oligonucleotidealso comprises at least one 3′ end with a single nucleotide overhang.

Following contacting the at least one target analyte with the at leastone probe, a one-ended adapter ligation method can further comprise: (2)providing a force to a location of the sample sufficient to release theidentifier oligonucleotide. In a non-limiting example, in aspects inwhich the probe comprises a photo-cleavable linker between theidentifier oligonucleotide and the target binding domain, aregion-of-interest (ROI) is excited with light of a sufficientwavelength capable of cleaving the photo-cleavable linker.

Following release of the identifier oligonucleotide, a one-ended adapterligation method can further comprise: (3) collecting the releasedidentifier oligonucleotide. By directing the force only to a specificlocation in step (2), identifier oligonucleotides are only released fromprobes within that location and not from probes located outside thatlocation. Thus, identifier oligonucleotides are collected only forprobes that are bound to targets within that location in step (3),thereby permitting detection of the identities and quantities of thetargets (proteins and/or nucleic acids) located only within thatlocation.

Following collection of the released identifier oligonucleotide, aone-ended adapter ligation method can further comprise: (4) ligating tothe released oligonucleotide collected in step (3) at least one nucleicacid adapter;

The nucleic acid adapter can comprise a nucleic acid sequence whichidentifies the specific location of the sample from which the identifieroligonucleotide was released. For example, if the identifieroligonucleotide was released from location of the sample designated “ROI#1”, the nucleic acid adapter would comprise a nucleic acid sequencethat corresponds to “ROI #1”. The nucleic acid adapter can also comprisea unique molecular identifier. The nucleic acid adapter can alsocomprise a second amplification primer binding site.

In some aspects, the nucleic acid adapter can also comprise a constantnucleic acid sequence to minimize ligation bias caused by differences insequences of particular identifier oligonucleotides. The constantnucleic acid sequence can comprise a cleavable moiety. The cleavablemoiety can be enzymatically cleavable. In a non-limiting example, theenzymatically cleavable moiety can be a USER sequence, wherein the USERsequence comprises the sequence GUGUATUG.

The nucleic acid adapter can comprise any combination of the featuresdescribed above.

The nucleic acid adapter can be a partially double-stranded nucleic acidmolecule. In aspects in which the nucleic acid adapter is partiallydouble-stranded, the nucleic acid adapter comprises a double-strandedannealed region and a single-stranded mismatched region.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a nucleic acid sequence which identifiesthe specific location of the sample from which the identifieroligonucleotide was released, the nucleic acid sequence which identifiesthe specific location of the sample from which the identifieroligonucleotide was released can be present in the double-strandedannealed region of the nucleic acid adapter.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a constant nucleic acid sequence tominimize ligation bias, the constant nucleic acid sequence to minimizeligation bias can be present in the double-stranded annealed region ofthe nucleic acid adapter.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a unique molecular identifier, the uniquemolecular identifier can be present in the single-stranded mismatchedregion.

In aspects in which the nucleic acid adapter is partiallydouble-stranded and comprises a second amplification primer bindingsite, the second amplification primer binding site can be present in thesingle-stranded mismatched region of the nucleic acid adapter.

After ligation of the at least one nucleic acid adapter, a two-endedadapter ligation method can further comprise: (5) amplifying theligation product produced in step (4); and (6) identifying the releasedoligonucleotide by sequencing the amplified products produced in step(5), thereby spatially detecting the at least one target analyte in thesample.

In other aspects of a one-ended adapter ligation method of the presentdisclosure, at least one of the amplification primers used in step (5)to amplify the ligation product produced in step (4) comprises a nucleicacid sequence which identifies the specific location of the tissuesample form which the identifier oligonucleotide was released. Forexample, if the identifier oligonucleotide was released from location ofthe sample designated “ROI #1”, at least one of the amplificationprimers would comprise a nucleic acid sequence that corresponds to “ROI#1”.

FIG. 2 shows a schematic of a preferred aspect of a one-ended adapterligation method of the present disclosure. In this aspect, the probecomprises a target binding domain that is an antibody that binds to atarget protein. In the upper left panel, the probe binds to the targetprotein. In upper right panel, the UV photo-cleavable linker locatedbetween the target binding domain and the identifier oligonucleotide iscleaved, releasing the identifier oligonucleotide. The identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target protein and a first amplification primer bindingsite. In this non-limiting example, the identifier oligonucleotide isdouble-stranded with one strand that comprises three separate nucleicacid molecules. The identifier oligonucleotide also comprises one 3′ endwith a single nucleotide overhang.

In the bottom panel of FIG. 2, a nucleic acid adapter is ligated to theend of the identifier oligonucleotide that comprises the 3′ singlenucleotide overhang. In this non-limiting example, the nucleic acidadapter is partially double-stranded and comprises a double-strandedannealed region and a single-stranded mismatched region. Present in thedouble-stranded annealed region is a constant nucleic acid sequence tominimize ligation bias and a nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released. Present in the single-stranded mismatchedregion is a unique molecular identifier and a second amplificationprimer binding site. Following ligation of the nucleic acid adapter tothe identifier oligonucleotide, the product is amplified usingamplification primers that bind to the first and the secondamplification primer binding sites and sequenced to identify the targetprotein bound by the probe.

In one aspect, the present disclosure provides a composition of anidentifier oligonucleotide ligated to one nucleic acid adapter forspatially detecting at least one target analyte in a sample.

An identifier oligonucleotide ligated to one nucleic acid adaptercomprises an identifier oligonucleotide, wherein the identifieroligonucleotide comprises a unique nucleic acid sequence which iscapable of identifying a target analyte in a sample and a firstamplification primer binding site. One end of the identifieroligonucleotide is attached to a nucleic acid adapter molecule, whereinthe nucleic acid adapter molecule is partially double-stranded andcomprises a double-stranded annealed region and a single-strandedmismatched region and a second single-stranded mismatched region. Thedouble-stranded mismatch region comprises a constant nucleic acidsequence to minimize ligation bias and a nucleic acid sequence nucleicacid sequence which is capable of identifying a specific location of asample. The single-stranded mismatched region comprises a secondamplification primer binding site. A schematic of an identifieroligonucleotide ligated to one nucleic acid adapter is shown in thebottom panel of FIG. 2.

In another aspect, the present disclosure provides compositions andmethods for spatially detecting at least one target analyte in a sampleusing the probes of the present disclosure in a method herein referredto as a “templated-primer extension method”.

A templated-primer extension method of the present disclosure cancomprise: (1) contacting at least one target analyte in a sample with atleast one probe of the present disclosure. The at least one targetanalyte can be a target protein or a target nucleic acid. In the aspectthat the at least one target analyte is a target protein, the probe cancomprise a target binding domain that is a target protein-binding regionthat can specifically bind to a target protein of interest. In theaspect that the at least one target analyte is a target nucleic acid,the probe can comprise a target nucleic acid-binding region that candirectly or indirectly hybridize to a target nucleic acid of interest.The probe further comprises an identifier oligonucleotide. Theidentifier oligonucleotide can comprise a unique nucleic acid sequencewhich identifies the target analyte bound to the target binding domain.The identifier oligonucleotide can also comprise a first amplificationprimer binding site.

Following contacting the at least one target analyte with the at leastone probe, a templated-primer extension method can further comprise: (2)providing a force to a location of the sample sufficient to release theidentifier oligonucleotide. In a non-limiting example, in aspects inwhich the probe comprises a photo-cleavable linker between theidentifier oligonucleotide and the target binding domain, aregion-of-interest (ROI) is excited with light of a sufficientwavelength capable of cleaving the photo-cleavable linker.

Following release of the identifier oligonucleotide, a templated-primerextension method can further comprise: (3) collecting the releasedidentifier oligonucleotide. By directing the force only to a specificlocation in step (2), identifier oligonucleotides are only released fromprobes within that location and not from probes located outside thatlocation. Thus, identifier oligonucleotides are collected only forprobes that are bound to targets within that location in step (3),thereby permitting detection of the identities and quantities of thetargets (proteins and/or nucleic acids) located only within thatlocation.

Following collection of the released identifier oligonucleotide, atemplated-primer extension method can further comprise: (4) hybridizingto the released identifier oligonucleotide collected in step (3) asingle stranded nucleic acid template.

The single stranded nucleic acid template can comprise a regioncomplementary to the unique nucleic acid sequence of the identifieroligonucleotide, thereby allowing for the hybridization of the singlestranded nucleic acid template and the collected identifieroligonucleotide.

The single stranded nucleic acid template can also comprise a nucleicacid sequence comprising a unique molecular identifier.

The single stranded nucleic acid template can also comprise a nucleicacid sequence which identifies the specific location of the sample fromwhich the identifier oligonucleotide was released.

The single stranded nucleic acid template can also comprise a nucleicacid sequence that is complementary to a second amplification primerbinding site.

The single stranded nucleic acid template can comprise any combinationof the features described above.

Following hybridization of the identifier oligonucleotide to the singlestranded nucleic acid template, a templated-primer extension method canfurther comprise: (5) extending the identifier oligonucleotide of step(4) to form an extension produce complementary to the single strandednucleic acid template, wherein the extension product comprises theidentifier oligonucleotide and the sequence complementary to the singlestranded nucleic acid template; (6) amplifying the extension product ofstep (6) using amplification primers that hybridize to the first andsecond amplification primer binding sites; and (7) identifying thereleased identifier oligonucleotide by sequencing the amplified productsproduced in step (6), thereby spatially detecting the at least onetarget analyte in the sample.

In some aspects, the single stranded nucleic acid template can comprisean affinity molecule. In aspects in which the single stranded nucleicacid template comprises an affinity molecule, a templated-primerextension method can further comprise an affinity purification stepbetween steps (4) and (5).

FIG. 3 shows a schematic of a preferred aspect of a templated-primerextension method of the present disclosure. In this aspect, the probecomprises a target binding domain that is an antibody that binds to atarget protein. In the upper left panel, the probe binds to the targetprotein. In upper right panel, the UV photo-cleavable linker locatedbetween the target binding domain and the identifier oligonucleotide iscleaved, releasing the identifier oligonucleotide. The identifieroligonucleotide comprises a unique nucleic acid sequence whichidentifies the target protein and a first amplification primer bindingsite. In the lower right panel, the identifier oligonucleotide ishybridized to a single stranded nucleic acid template. In thisnon-limiting example, the single-stranded nucleic acid templatecomprises an affinity molecule, a nucleic acid sequence complementary tothe unique nucleic acid sequence of the identifier oligonucleotide, afirst unique molecular identifier, and a sequence complementary to asecond amplification primer biding site. The identifier oligonucleotideis extended to form an extension product complementary to the singlestranded nucleic acid template. As shown in the lower left panel, theextension product comprises the identifier oligonucleotide, the nucleicacid sequence complementary to the first unique molecular identifier,and the second amplification primer binding site. Following theextension reaction, the primer extension product is amplified usingamplification primers that bind to the first and the secondamplification primer binding sites. In this non-limiting example, one ofthe amplification primers comprises a nucleic acid sequence whichidentifies the specific location of the sample from which the identifieroligonucleotide was released. The amplified product is then sequenced toidentify the target protein bound by the probe.

In one aspect, the present disclosure provides a composition of atemplate-extended identifier oligonucleotide for spatially detecting atleast one target analyte in a sample. The template-extended identifieroligonucleotide comprises a first flow cell adapter sequence suitablefor sequencing, followed by a first unique molecular identifier,followed by an identifier oligonucleotide, followed by a second uniquemolecular identifier, followed by a second amplification primer bindingsite, followed by a third unique molecular identifier, followed by asecond flow cell adapter sequence suitable for sequencing. Theidentifier oligonucleotide comprises a first amplification primerbinding site and a unique nucleic acid sequence which is capable ofidentifying a target analyte in a sample. A schematic of atemplate-extended identifier oligonucleotide is shown in the bottompanel of FIG. 4.

In another aspect, the present disclosure provides compositions andmethods for spatially detecting at least one target analyte in a sampleusing the probes of the present disclosure in a method herein referredto as a “short probe hybridization method”.

A short probe hybridization method of the present disclosure cancomprise: (1) contacting at least one target analyte in a sample with atleast one probe of the present disclosure. The at least one targetanalyte can be a target protein or a target nucleic acid. In the aspectthat the at least one target analyte is a target protein, the probe cancomprise a target binding domain that is a target protein-binding regionthat can specifically bind to a target protein of interest. In theaspect that the at least one target analyte is a target nucleic acid,the probe can comprise a target nucleic acid-binding region that candirectly or indirectly hybridize to a target nucleic acid of interest.The probe further comprises an identifier oligonucleotide. Theidentifier oligonucleotide can comprise a unique nucleic acid sequencewhich identifies the target analyte bound to the target binding domain.

Following contacting the at least one target analyte with the at leastone probe, a short probe hybridization method can further comprise: (2)providing a force to a location of the sample sufficient to release theidentifier oligonucleotide. In a non-limiting example, in aspects inwhich the probe comprises a photo-cleavable linker between theidentifier oligonucleotide and the target binding domain, aregion-of-interest (ROI) is excited with light of a sufficientwavelength capable of cleaving the photo-cleavable linker.

Following release of the identifier oligonucleotide, a short probehybridization method can further comprise: (3) collecting the releasedidentifier oligonucleotide. By directing the force only to a specificlocation in step (2), identifier oligonucleotides are only released fromprobes within that location and not from probes located outside thatlocation. Thus, identifier oligonucleotides are collected only forprobes that are bound to targets within that location in step (3),thereby permitting detection of the identities and quantities of thetargets (proteins and/or nucleic acids) located only within thatlocation.

Following collection of the released identifier oligonucleotide, a shortprobe hybridization method can further comprise: (4) hybridizing to therelease identifier oligonucleotide a first nucleic acid probe and asecond nucleic acid probe.

The first or the second nucleic acid probe can comprise a nucleic acidsequence complementary to a portion of the identifier oligonucleotide.The first or the second nucleic acid probe can also comprise a nucleicacid sequence which identifies the specific location of the sample fromwhich the identifier oligonucleotide was released. The first or thesecond nucleic acid probe can also comprise a nucleic acid sequencecomprising unique molecular identifier. The first nucleic acid probe cancomprise a first amplification primer binding site. The second nucleicacid probe can comprise a second amplification primer binding site.

The first or the second nucleic acid probe can comprise any combinationof the features described above. In a preferred aspect depicted in FIG.5, the first nucleic acid probe comprises a first amplification primerbinding site, a nucleic acid sequence which identifies the specificlocation of the sample from which the identifier oligonucleotide wasreleased and a nucleic acid sequence complementary to the identifieroligonucleotide. In the same preferred aspect, the second nucleic acidprobe comprises a second amplification primer binding site, a nucleicacid sequence comprising a unique molecular identifier and a nucleicacid sequence complementary to a portion of the identifieroligonucleotide. In this preferred aspect, the nucleic acid sequencewhich identifies the specific location of the sample from which theidentifier oligonucleotide was released is located 5′ to the firstamplification primer binding site and the unique molecular identifier islocated 3′ to the second amplification primer binding site.

In another preferred aspect depicted in FIG. 6, the first nucleic acidprobe comprises a first amplification primer binding site, a nucleicacid sequence which identifies the specific location of the sample fromwhich the identifier oligonucleotide was released, a nucleic acidsequence comprising a unique molecular identifier and a nucleic acidsequence complementary to a portion of the identifier oligonucleotide.In this same preferred aspect, the second nucleic acid probe comprises asecond amplification primer binding site and a nucleic acid sequencecomplementary to the identifier oligonucleotide. In this preferredaspect, the nucleic acid sequence which identifies the specific locationof the sample from which the identifier oligonucleotide was released andthe unique molecular identifier are located 5′ to the firstamplification primer binding site.

In another preferred aspect depicted in FIG. 7, the first nucleic acidprobe comprises a first amplification primer binding site and a nucleicacid sequence complementary to a portion of the identifieroligonucleotide. In this same preferred aspect, the second nucleic acidprobe comprises a second amplification primer binding site, a nucleicacid sequence which identifies the specific location of the sample fromwhich the identifier oligonucleotide was released, a nucleic acidsequence comprising a unique molecular identifier and a nucleic acidsequence complementary to the identifier oligonucleotide. In thispreferred aspect, the nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released and the unique molecular identifier arelocated 3′ to the second amplification primer binding site.

In another preferred aspect depicted in FIG. 15, the first nucleic acidprobe comprises a first amplification primer binding site, a nucleicacid sequence comprising a unique molecular identifier and a nucleicacid sequence complementary to a portion of the identifieroligonucleotide. In the same preferred aspect, the second nucleic acidprobe comprises a second amplification primer binding site and a nucleicacid sequence complementary to a portion of the identifieroligonucleotide. In this preferred aspect, the nucleic acid sequencecomprising a unique molecular identifier is located 3′ to the firstamplification binding site.

In another preferred aspect depicted in FIG. 17, the first nucleic acidprobe comprises a first amplification primer binding site, a nucleicacid sequence comprising a unique molecular identifier and a nucleicacid sequence complementary to a portion of the identifieroligonucleotide. In the same preferred aspect, the second nucleic acidprobe comprises a second amplification primer binding site and a nucleicacid sequence complementary to a portion of the identifieroligonucleotide. In this preferred aspect, the nucleic acid sequencecomprising a unique molecular identifier is located 5′ to the firstamplification binding site.

The first nucleic acid probe and the second nucleic acid probe canhybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are adjacent and are not overlapping.Alternatively, the first nucleic acid probe and the second nucleic acidprobe can hybridize to the identifier oligonucleotide such that thefirst and the second nucleic acid probes are not adjacent and are notoverlapping.

Following hybridization of the first and the second nucleic acid probeto the identifier oligonucleotide, a short probe hybridization methodcan further comprise: (5) in the aspect in which the first and thesecond nucleic acid probe hybridize to the identifier oligonucleotidesuch that the first and the second nucleic acid probes are adjacent andare not overlapping, ligating the first and the second nucleic acidprobes together, for example, by performing a nick repair reaction.Alternatively, in the aspect in which the first and the second nucleicacid probe hybridize to the identifier oligonucleotide such that thefirst and the second nucleic acid probes are not adjacent and are notoverlapping, the method comprises ligating the first and the secondnucleic acid probes together, for example, by performing a gap extensionreaction and a nick repair reaction, such that the first and the secondnucleic acid probes are ligated together.

Following ligation of the first and the second nucleic acid probe, ashort probe hybridization method can further comprise: (6) amplifyingthe ligation product produced in step (5) using amplification primersthat hybridize to the first and second amplification primer bindingsites; and (7) identifying the released identifier oligonucleotide bysequencing the amplified products produced in step (6), therebyspatially detecting the at least one target analyte in the sample.

In one aspect, the present disclosure provides a composition of anidentifier oligonucleotide-short nucleic acid probe complex forspatially detecting at least one target analyte in a sample. Anidentifier oligonucleotide-short nucleic acid probe complex comprises anidentifier oligonucleotide hybridized to a first nucleic acid probe anda second nucleic acid probe. The identifier oligonucleotide comprises aunique nucleic acid sequence capable of identifying a target analyte ina sample. The first nucleic acid probe comprises a first amplificationprimer binding site, followed by a unique nucleic acid sequence capableof identifying a specific location in a sample, followed by a regioncomplementary to the identifier oligonucleotide. The second nucleic acidprobe comprises a second amplification primer binding site, followed bya nucleic acid sequence comprising a unique molecular identifier,followed by a region complementary to the identifier oligonucleotide. Aschematic of an identifier oligonucleotide-short nucleic acid probecomplex is depicted in FIG. 5.

In one aspect, the present disclosure provides a composition of anidentifier oligonucleotide-short nucleic acid probe complex forspatially detecting at least one target analyte in a sample. Anidentifier oligonucleotide-short nucleic acid probe complex comprises anidentifier oligonucleotide hybridized to a first nucleic acid probe anda second nucleic acid probe. The identifier oligonucleotide comprises aunique nucleic acid sequence capable of identifying a target analyte ina sample. The first nucleic acid probe comprises a first amplificationprimer binding site, followed by a nucleic acid sequence comprising aunique molecular identifier, followed by a region complementary to theidentifier oligonucleotide, wherein the nucleic acid sequence comprisinga unique molecular identifier is located 3′ to the first amplificationprimer binding site. The second nucleic acid probe comprises a secondamplification primer binding site followed by a region complementary tothe identifier oligonucleotide. A schematic of an identifieroligonucleotide-short nucleic acid probe complex is depicted in FIG. 15.

In one aspect, the present disclosure provides a composition of anidentifier oligonucleotide-short nucleic acid probe complex forspatially detecting at least one target analyte in a sample. Anidentifier oligonucleotide-short nucleic acid probe complex comprises anidentifier oligonucleotide hybridized to a first nucleic acid probe anda second nucleic acid probe. The identifier oligonucleotide comprises aunique nucleic acid sequence capable of identifying a target analyte ina sample. The first nucleic acid probe comprises a first amplificationprimer binding site, followed by a nucleic acid sequence comprising aunique molecular identifier, followed by a region complementary to theidentifier oligonucleotide, wherein the nucleic acid sequence comprisinga unique molecular identifier is located 5′ to the first amplificationprimer binding site. The second nucleic acid probe comprises a secondamplification primer binding site followed by a region complementary tothe identifier oligonucleotide. A schematic of an identifieroligonucleotide-short nucleic acid probe complex is depicted in FIG. 17.

FIG. 16 shows a schematic overview of an exemplary short probehybridization method of the present disclosure. First at least onetarget analyte in a sample is contacted with at least one probe of thepresent disclosure. The at least one target analyte can be a targetprotein or a target nucleic acid. In the aspect that the at least onetarget analyte is a target protein, the probe can comprise a targetbinding domain that is a target protein-binding region that canspecifically bind to a target protein of interest. In the aspect thatthe at least one target analyte is a target nucleic acid, the probe cancomprise a target nucleic acid-binding region that can directly orindirectly hybridize to a target nucleic acid of interest. The probefurther comprises an identifier oligonucleotide. The identifieroligonucleotide can comprise a unique nucleic acid sequence whichidentifies the target analyte bound to the target domain.

Following contacting the at least one target analyte with the at leastone probe, a force is then provided to a location of the samplesufficient to release the identifier oligonucleotide. The identifieroligonucleotide is collected following release, as shown in the toppanel of FIG. 16.

As shown in the second panel from the top of FIG. 16, the releasedidentifier oligonucleotide is then hybridized to a first nucleic acidprobe and a second nucleic acid probe. In this non-limiting example, thefirst nucleic acid probe comprises a first amplification primer bindingsite, a nucleic acid sequence comprising a unique molecular identifierand a nucleic acid sequence complementary to a portion of the identifieroligonucleotide. The nucleic acid sequence comprising the uniquemolecular identifier is located 3′ to the first amplification primerbinding site. The second nucleic acid probe comprises a secondamplification primer binding site and a nucleic acid sequencecomplementary to a portion of the identifier oligonucleotide. In thisnon-limiting example, the first and the second nucleic acid probehybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are adjacent and are not overlapping.Following hybridization to the identifier oligonucleotide, the first andsecond probe are ligated together, for example, by performing a nickrepair reaction.

Following ligation of the first and second nucleic acid probes, theligation product is amplified via PCR using amplification primers thathybridize to the first and second amplification primer binding sites. Asshown in the second panel from the bottom of FIG. 16, the amplificationprimer that hybridizes to the second amplification primer binding sitecomprises a first flow cell binding site, a first nucleic acid sequencewhich identifies the specific location of the sample from which theidentifier oligonucleotide was released and a nucleic acid sequencecomplementary to the second amplification primer binding site. Theamplification primer that hybridizes to the first amplification primerbinding site comprises a second flow cell binding site, a second nucleicacid sequence which identifies the specific location from which theidentifier oligonucleotide was released and a nucleic acid sequencecomplementary to the first amplification primer binding site. The PCRproduct shown in the bottom panel of FIG. 16 is then sequenced toidentify the released oligonucleotide, thereby spatially detecting theat least one target analyte in the sample.

FIG. 18 shows a schematic overview of an exemplary short probehybridization method of the present disclosure. First, at least onetarget analyte in a sample is contacted with at least one probe of thepresent disclosure. The at least one target analyte can be a targetprotein or a target nucleic acid. In the aspect that the at least onetarget analyte is a target protein, the probe can comprise a targetbinding domain that is a target protein-binding region that canspecifically bind to a target protein of interest. In the aspect thatthe at least one target analyte is a target nucleic acid, the probe cancomprise a target nucleic acid-binding region that can directly orindirectly hybridize to a target nucleic acid of interest. The probefurther comprises an identifier oligonucleotide. The identifieroligonucleotide can comprise a unique nucleic acid sequence whichidentifies the target analyte bound to the target domain.

Following contacting the at least one target analyte with the at leastone probe, a force is then provided to a location of the samplesufficient to release the identifier oligonucleotide. The identifieroligonucleotide is collected following release, as shown in the toppanel of FIG. 18.

As shown in the second panel from the top of FIG. 18, the releasedidentifier oligonucleotide is then hybridized to a first nucleic acidprobe and a second nucleic acid probe. In this non-limiting example, thefirst nucleic acid probe comprises a first amplification primer bindingsite, a nucleic acid sequence comprising a unique molecular identifierand a nucleic acid sequence complementary to a portion of the identifieroligonucleotide. The nucleic acid sequence comprising the uniquemolecular identifier is located 5′ to the first amplification primerbinding site. The second nucleic acid probe comprises a secondamplification primer binding site and a nucleic acid sequencecomplementary to a portion of the identifier oligonucleotide. In thisnon-limiting example, the first and the second nucleic acid probehybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are adjacent and are not overlapping.Following hybridization to the identifier oligonucleotide, the first andsecond probe are ligated together, for example, by performing a nickrepair reaction.

Following ligation of the first and second nucleic acid probes, theligation product is amplified via PCR using amplification primers thathybridize to the first and second amplification primer binding sites. Asshown in the second panel from the bottom of FIG. 18, the amplificationprimer that hybridizes to the second amplification primer binding sitecomprises a first flow cell binding site, a first nucleic acid sequencewhich identifies the specific location of the sample from which theidentifier oligonucleotide was released and a nucleic acid sequencecomplementary to the second amplification primer binding site. Theamplification primer that hybridizes to the first amplification primerbinding site comprises a second flow cell binding site, a second nucleicacid sequence which identifies the specific location from which theidentifier oligonucleotide was released and a nucleic acid sequencecomplementary to the first amplification primer binding site. The PCRproduct shown in the bottom panel FIG. 18 is then sequenced to identifythe released oligonucleotide, thereby spatially detecting the at leastone target analyte in the sample.

In another aspect, the present disclosure provides compositions andmethods for spatially detecting at least one target analyte in a sampleusing the probes of the present disclosure in a method herein referredto as a “long probe hybridization method”.

A long probe hybridization method of the present disclosure cancomprise: (1) contacting at least one target analyte in a sample with atleast one probe of the present disclosure. The at least one targetanalyte can be a target protein or a target nucleic acid. In the aspectthat the at least one target analyte is a target protein, the probe cancomprise a target binding domain that is a target protein-binding regionthat can specifically bind to a target protein of interest. In theaspect that the at least one target analyte is a target nucleic acid,the probe can comprise a target nucleic acid-binding region that candirectly or indirectly hybridize to a target nucleic acid of interest.The probe further comprises an identifier oligonucleotide. Theidentifier oligonucleotide can comprise a unique nucleic acid sequencewhich identifies the target analyte bound to the target binding domain.

Following contacting the at least one target analyte with the at leastone probe, a long probe hybridization method can further comprise: (2)providing a force to a location of the sample sufficient to release theidentifier oligonucleotide. In a non-limiting example, in aspects inwhich the probe comprises a photo-cleavable linker between theidentifier oligonucleotide and the target binding domain, aregion-of-interest (ROI) is excited with light of a sufficientwavelength capable of cleaving the photo-cleavable linker.

Following release of the identifier oligonucleotide, a long probehybridization method can further comprise: (3) collecting the releasedidentifier oligonucleotide. By directing the force only to a specificlocation in step (2), identifier oligonucleotides are only released fromprobes within that location and not from probes located outside thatlocation. Thus, identifier oligonucleotides are collected only forprobes that are bound to targets within that location in step (3),thereby permitting detection of the identities and quantities of thetargets (proteins and/or nucleic acids) located only within thatlocation.

Following collection of the released identifier oligonucleotide(s), along probe hybridization method can further comprise: (4) hybridizing tothe released identifier oligonucleotide a first nucleic acid probe and asecond nucleic acid probe.

The first or the second nucleic acid probe can comprise a nucleic acidsequence complementary to a portion of the identifier oligonucleotide.The first or the second nucleic acid probe can also comprise a nucleicacid sequence which identifies the specific location of the sample fromwhich the identifier oligonucleotide was released.

The first or the second nucleic acid probe can also comprise a firstunique molecular identifier. The first or the second nucleic acid probecan also comprise a second unique molecular identifier. The first or thesecond nucleic acid probe can also comprise a third unique molecularidentifier.

The first nucleic acid probe can comprise a first amplification primerbinding site.

The first nucleic acid probe can also comprise a first flow cell bindingsite. The second nucleic acid probe can comprise a second flow cellbinding site.

The first and the second nucleic acid probes can comprise anycombination of the features described above. In a preferred aspectdepicted in FIG. 8, the first nucleic acid probe comprises a first flowcell binding site, a first unique molecular identifier, a firstamplification primer binding site, a nucleic acid sequence whichidentifies the specific location of the sample from which the identifieroligonucleotide was released and a nucleic acid sequence complementaryto the identifier oligonucleotide. In the same preferred aspect, thesecond nucleic acid probe comprises a second flow cell binding site, asecond unique molecular identifier, a third unique molecular identifierand a nucleic acid sequence complementary to the identifieroligonucleotide. In this preferred aspect, the nucleic acid sequencewhich identifies the specific location of the sample from which theidentifier oligonucleotide was released and the first unique molecularidentifier are located 5′ to the first flow cell binding site and thesecond and the third unique molecular identifiers are located 3′ to thesecond flow cell binding site.

In another preferred aspect depicted in FIG. 9, the first nucleic acidprobe comprises a first flow cell binding site, a first unique molecularidentifier, a second unique molecular identifier, a first amplificationprimer binding site, a nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released and a nucleic acid sequence complementaryto the identifier oligonucleotide. In the same preferred aspect, thesecond nucleic acid probe comprises a second flow cell binding site, athird unique molecular identifier and a nucleic acid sequencecomplementary to the identifier oligonucleotide. In this preferredaspect, the first unique molecular identifier, the second uniquemolecular identifier and the nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released are located 5′ to the first flow cellbinding site and the third unique molecular identifier is located 3′ tothe second flow cell binding site.

In another preferred aspect depicted in FIG. 10, the first nucleic acidprobe comprises a first flow cell binding site, a first unique molecularidentifier, a first amplification primer binding site and a nucleic acidsequence complementary to the identifier oligonucleotide. In this samepreferred aspect, the second nucleic acid probe comprises a second flowcell binding site, a second unique molecular identifier, a third uniquemolecular identifier, a nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released and a nucleic acid sequence complementaryto the identifier oligonucleotide. In this preferred aspect, the firstunique molecular identifier is located 5′ to the first flow cell bindingsite and the second unique molecular identifier, the third uniquemolecular identifier, and the nucleic acid sequence which identifies thespecific location of the sample form which the identifieroligonucleotide was released are located 3′ to the second amplificationprimer binding site.

The first nucleic acid probe and the second nucleic acid probe canhybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are adjacent and are not overlapping.Alternatively, the first nucleic acid probe and the second nucleic acidprobe can hybridize to the identifier oligonucleotide such that thefirst and the second nucleic acid probes are not adjacent and are notoverlapping.

Following hybridization of the first and the second nucleic acid probeto the identifier oligonucleotide, a long probe hybridization method canfurther comprise: (5) in the aspect in which the first and the secondnucleic acid probe hybridize to the identifier oligonucleotide such thatthe first and the second nucleic acid probes are adjacent and are notoverlapping, performing a nick repair reaction such that the first andthe second nucleic acid probes are ligated together. Alternatively, inthe aspect in which the first and the second nucleic acid probehybridize to the identifier oligonucleotide such that the first and thesecond nucleic acid probes are not adjacent and are not overlapping, themethod comprises performing a gap extension and a nick repair reactionsuch that the first and the second nucleic acid probes are ligatedtogether.

The name method can further comprise: (6) amplifying the ligationproduct produced in step (5) using amplification primers that hybridizeto the first and second amplification primer binding sites; and (7)identifying the released identifier oligonucleotide by sequencing theamplified products produced in step (6), thereby spatially detecting theat least one target analyte in the sample.

In one aspect, the present disclosure provides a composition of anidentifier oligonucleotide-long nucleic acid probe complex for spatiallydetecting at least one target analyte in a sample. An identifieroligonucleotide-long nucleic acid probe complex comprises an identifieroligonucleotide hybridized to a first nucleic acid probe and a secondnucleic acid probe. The identifier oligonucleotide comprises a uniquenucleic acid sequence capable of identifying a target analyte in asample. The first nucleic acid probe comprises a first flow cell bindingsite suitable for sequencing, followed by a first unique molecularidentifier, followed by a first amplification primer binding site,followed by a unique nucleic acid sequence capable of identifying aspecific location in a sample, followed by a region complementary to theidentifier oligonucleotide. The second nucleic acid probe comprises asecond flow cell binding site, followed by a second unique molecularidentifier, followed by a third unique molecular identifier, followed bya region complementary to the identifier oligonucleotide. A schematic ofan identifier oligonucleotide-short nucleic acid probe complex isdepicted in FIG. 8.

In another aspect, the present disclosure provides compositions andmethods for spatially detecting at least one target analyte in a sampleusing the probes of the present disclosure in a method herein referredto as a “direct PCR method”.

A direct PCR method of the present disclosure can comprise: (1)contacting at least one target analyte in a sample with at least oneprobe of the present disclosure. The at least one target analyte can bea target protein or a target nucleic acid. In the aspect that the atleast one target analyte is a target protein, the probe can comprise atarget binding domain that is a target protein-binding region that canspecifically bind to a target protein of interest. In the aspect thatthe at least one target analyte is a target nucleic acid, the probe cancomprise a target nucleic acid-binding region that can directly orindirectly hybridize to a target nucleic acid of interest. The probefurther comprises an identifier oligonucleotide. The identifieroligonucleotide can comprise a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain. Theidentifier oligonucleotide can also comprise a first amplificationprimer binding site, a second amplification primer binding site, or aunique molecular identifier. The identifier oligonucleotide can compriseany combination of these features. Any of these features can also beflanked by regions comprising constant nucleic acid sequences of about 1nucleotide to about 10 nucleotides.

Following contacting the at least one analyte with the at least oneprobe, a direct PCR method further comprises: (2) providing a force to alocation of the sample sufficient to release the identifieroligonucleotide. In a non-limiting example, in aspects in which theprobe comprises a photo-cleavable linker between the identifieroligonucleotide and the target binding domain, a region-of-interest(ROI) is excited with light of a sufficient wavelength capable ofcleaving the photo-cleavable linker.

A direct PCR method can further comprise: (3) collecting the releasedidentifier oligonucleotide. By directing the force only to a specificlocation in step (2), identifier oligonucleotides are only released fromprobes within that location and not from probes located outside thatlocation. Thus, identifier oligonucleotides are collected only forprobes that are bound to targets within that location in step (3),thereby permitting detection of the identities and quantities of thetargets (proteins and/or nucleic acids) located only within thatlocation.

Following release of the identifier oligonucleotide, a direct PCR methodcan further comprise: (4) amplifying the released identifieroligonucleotide using a first amplification primer capable of binding tothe first amplification primer binding site and a second amplificationprimer capable of binding to the second amplification primer bindingsite. In some aspects, at least one of the amplification primerscomprises a nucleic acid sequence which identifies the specific locationof the sample from which the identifier oligonucleotide was released.For example, if the identifier oligonucleotide was released fromlocation of the sample designated “ROI #1”, at least one of theamplification primers would comprise a nucleic acid sequence thatcorresponds to “ROI #1”. In still other aspects, at least one of theamplification primers comprises a unique molecular identifier.

Following amplification, a direct PCR method of the present disclosurecan further comprise: (5) identifying the released oligonucleotide bysequencing the amplified products produced in step (5), therebyspatially detecting the at least one target analyte in the sample.

FIG. 11 shows a schematic of a preferred aspect of a direct PCR methodof the present disclosure. In this aspect, the probe comprises a targetbinding domain comprising a nucleic acid sequence that is complementaryto a target nucleic acid. In the upper panel, the probe hybridizes tothe target nucleic acid. In the lower panel, a UV photo-cleavable linkerlocated between the target binding domain and the identifieroligonucleotide is cleaved, releasing the identifier oligonucleotide.The identifier oligonucleotide comprises a first amplification primerbinding site, a second amplification primer binding site, a uniquemolecular identifier, and a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain.Located between these four features are constant spacer regions that are3 nucleotides in length. The identifier oligonucleotide isdouble-stranded and comprises a strand that comprises 3 separate nucleicacid molecules. After release, the identifier oligonucleotide isamplified using a first amplification primer that hybridizes to thefirst amplification primer binding site and comprises a nucleic acidsequence which identifies the specific location of the sample from whichthe identifier oligonucleotide was released and a second amplificationprimer that hybridizes to the second amplification primer binding site.The amplified product is then sequenced to identify the target nucleicacid bound by the probe.

FIG. 19 shows a schematic of a preferred aspect of a direct PCR methodof the present disclosure. In this aspect, the identifieroligonucleotide comprises a first amplification primer binding site, anucleic acid sequence comprising a unique molecular identifier, a uniquenucleic acid sequence which identifies the target analyte bound to thetarget binding domain and a second amplification primer binding site, asshown in the top panel of FIG. 19. The identifier oligonucleotide isamplified using amplification primers that hybridize to the first andsecond amplification primer binding sites. As shown in the middle panelof FIG. 19, the amplification primer that hybridizes to the secondamplification primer binding site comprises a first flow cell bindingsite, a first nucleic acid sequence which identifies the specificlocation of the sample from which the identifier oligonucleotide wasreleased and a nucleic acid sequence complementary to the secondamplification primer binding site. The amplification primer thathybridizes to the first amplification primer binding site comprises asecond flow cell binding site, a second nucleic acid sequence whichidentifies the specific location of the sample from which the identifieroligonucleotide was released and a nucleic acid sequence complementaryto the first amplification primer binding site. The PCR product shown inthe bottom panel of FIG. 19 is sequenced to identify the releasedoligonucleotide, thereby spatially detecting the at least one targetanalyte in the sample.

In one aspect, the present disclosure provides a composition of adirect-PCR compatible identifier oligonucleotide for spatially detectingat least one target analyte in a sample. A direct-PCR compatibleidentifier oligonucleotide comprises a first amplification primerbinding site, followed a unique nucleic acid sequence which is capableof identifying a target analyte in a sample, followed by a uniquemolecular identifier, followed by a second amplification primer bindingsite. A schematic of a direct-PCR compatible identifier oligonucleotideis depicted in the lower panel of FIG. 11.

In one aspect, the present disclosure provides a composition of adirect-PCR compatible identifier oligonucleotide for spatially detectingat least one target analyte in a sample. A direct-PCR compatibleidentifier oligonucleotide comprises a first amplification primerbinding site, followed a nucleic acid sequence comprising a uniquemolecular identifier, followed by a unique nucleic acid sequence whichis capable of identifying a target analyte in a sample, followed by asecond amplification primer binding site. A schematic of a direct-PCRcompatible identifier oligonucleotide is shown in the top panel of FIG.19.

In some aspects of the methods of the present disclosure at least one,or at least two, or at least three, or at least four, or at least five,or at least six, or at least seven, or at least eight, or at least nine,or at least ten, or at least eleven, or at least twelve, or at leastthirteen, or at least fourteen, or at least fifteen, or at leastsixteen, or at least seventeen, or at least eighteen, or at leastnineteen, or at least twenty, or at least thirty, or at least forty, orat least fifty, or at least sixty, or at least seventy, or at leasteighty, or at least ninety, or at least one hundred probes can been to asingle target analyte. As used herein, the term “tiling” is used todescribe when more than one probe of the present disclosure is bound toa target analyte. The top panel of FIG. 27 shows the tiling of probesonto a target RNA. Tiling multiple probes onto a target analyte meansthat each target analyte will be individually detected multiple times,increasing the overall accuracy of the measurement. In a non-limitingexample, as shown in the bottom panel of FIG. 27, in the case where 10probes are tiled onto a single target RNA, one of the probes may beincorrectly detected too many times (outlier high count probe), whileanother probe may be incorrectly detected too few times (outlier lowcount probe). However, the other 8 probes may be detected at a similarlevel, indicating that the two outliers should be discarded duringanalysis and the signals from the 8 probes used to generate a moreaccurate measurement of the abundance of the target RNA.

The present disclosure provides compositions and methods for spatiallydetecting at least one target analyte in a sample using the probes ofthe present disclosure in a method herein referred to as an “enzyme freemethod”.

An enzyme free method of the present disclosure can comprise: (1)contacting at least one target analyte in a sample with at least oneprobe of the present disclosure. The at least one target analyte can bea target protein or a target nucleic acid. In the aspect that the atleast one target analyte is a target protein, the probe can comprise atarget binding domain that is a target protein-binding region that canspecifically bind to a target protein of interest. In the aspect thatthe at least one target analyte is a target nucleic acid, the probe cancomprise a target nucleic acid-binding region that can directly orindirectly hybridize to a target nucleic acid of interest. The probefurther comprises an identifier oligonucleotide. An identifieroligonucleotide can comprise a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain. Anidentifier oligonucleotide can also comprise a capture probe bindingsite.

Following contacting at least one target analyte with at least oneprobe, an enzyme free method can further comprise: (2) providing a forceto a location of the sample sufficient to release the identifieroligonucleotide. In a non-limiting example, in aspects in which theprobe comprises a photo-cleavable linker between the identifieroligonucleotide and the target binding domain, a region-of-interest(ROI) is excited with light of a sufficient wavelength capable ofcleaving the photo-cleavable linker.

Following release of an identifier oligonucleotide, an enzyme freemethod can further comprise: (3) collecting the released identifieroligonucleotide. By directing the force only to a specific location instep (2), identifier oligonucleotides are only released from probeswithin that location and not from probes located outside that location.Thus, identifier oligonucleotides are collected only for probes that arebound to targets within that location in step (3), thereby permittingdetection of the identities and quantities of the targets (proteinsand/or nucleic acids) located only within that location.

Following collection of a released identifier oligonucleotide, an enzymefree method can further comprise: (4) hybridizing to a releasedidentifier oligonucleotide a capture probe.

A capture probe can comprise a region complementary to the capture probebinding site. A capture probe can also comprise an affinity molecule.

Following hybridization of a capture probe, an enzyme free method canfurther comprise: (5) Identifying a released identifier oligonucleotideby sequencing the hybridized product produced in step (4), therebyspatially detecting the at least one target analyte in the at least onecell in a tissue sample.

A hybridized product produced in step (4) can be sequenced using anenzyme free method of sequencing. Enzyme-free methods of sequencing havebeen described in, e.g., US2014946386 and U.S. Ser. No. 15/819,151 (U.S.Pat. No. 10,415,080), each of which is incorporated herein by referencein its entirety.

FIG. 12 shows a schematic of a preferred aspect of an enzyme free methodof the present disclosure. In this aspect, the probe comprises a targetbinding domain comprising a nucleic acid sequence that is complementaryto a target nucleic acid. In the top panel, the probe hybridizes to thetarget nucleic acid. In the middle panel, a UV photo-cleavable linkerlocated between the target binding domain and the identifieroligonucleotide is cleaved, releasing the identifier oligonucleotide.The identifier oligonucleotide comprises a unique nucleic acid sequencewhich identifies the target nucleic acid bound to the target bindingdomain and a capture probe binding site. After release, the identifieroligonucleotide is hybridized to a capture probe, as depicted in thebottom panel. The capture probe comprises a nucleic acid sequencecomplementary to the capture probe binding site and an affinitymolecule. The hybridized product is then sequenced using enzyme freesequencing methods to identify the target nucleic acid bound by theprobe.

In one aspect, the present disclosure provides a composition of ahybridized identifier oligonucleotide-capture probe complex forspatially detecting at least one target analyte in a sample. Ahybridized identifier oligonucleotide-capture probe complex comprises anidentifier oligonucleotide hybridized to a capture probe. An identifieroligonucleotide comprises a unique nucleic acid sequence capable ofidentifying a specific a target analyte in a sample and a capture probebinding site. A capture probe comprises an affinity molecule and aregion complementary to the capture probe binding site. A schematic of ahybridized identifier oligonucleotide-capture probe complex is depictedin the bottom of panel FIG. 12.

The present disclosure provides compositions and methods for spatiallydetecting at least one target analyte in a sample using the probes ofthe present disclosure in a method herein referred to as a “multiplexedenzyme free method”.

A multiplexed enzyme free method of the present disclosure can comprise:(1) contacting at least one target analyte in a sample with at least oneprobe of the present disclosure. The at least one target analyte can bea target protein or a target nucleic acid. In the aspect that the atleast one target analyte is a target protein, the probe can comprise atarget binding domain that is a target protein-binding region that canspecifically bind to a target protein of interest. In the aspect thatthe at least one target analyte is a target nucleic acid, the probe cancomprise a target nucleic acid-binding region that can directly orindirectly hybridize to a target nucleic acid of interest. The probefurther comprises an identifier oligonucleotide. The identifieroligonucleotide can comprise a unique nucleic acid sequence whichidentifies the target analyte bound to the target binding domain. Theidentifier oligonucleotide can also comprise a capture probe bindingsite. The identifier oligonucleotide can also comprise a multiplexingprobe binding site.

Following contacting the at least one target analyte with the at leastone probe, a multiplexed enzyme free method can further comprise: (2)providing a force to a location of the sample sufficient to release theidentifier oligonucleotide. In a non-limiting example, in aspects inwhich the probe comprises a photo-cleavable linker between theidentifier oligonucleotide and the target binding domain, aregion-of-interest (ROI) is excited with light of a sufficientwavelength capable of cleaving the photo-cleavable linker.

Following release of the identifier oligonucleotide, a multiplexedenzyme free method can further comprise: (3) collecting the releasedidentifier oligonucleotide. By directing the force only to a specificlocation in step (2), identifier oligonucleotides are only released fromprobes within that location and not from probes located outside thatlocation. Thus, identifier oligonucleotides are collected only forprobes that are bound to targets within that location in step (3),thereby permitting detection of the identities and quantities of thetargets (proteins and/or nucleic acids) located only within thatlocation.

Following collection of the released identifier oligonucleotide, amultiplexed enzyme free method can further comprise: (4) hybridizing tothe released identifier oligonucleotide a capture probe and amultiplexing probe.

A capture probe can comprise a region complementary to the capture probebinding site. A capture probe can also comprise an affinity molecule.

A multiplexing probe can comprise a region complementary to themultiplexing probe binding site. A multiplexing probe can also comprisea nucleic acid sequence which identifies the specific location of thesample from which the identifier oligonucleotide was released and aregion complementary to the multiplexing probe binding site.

Following hybridization of a capture probe and a multiplexing probe, amultiplexed enzyme free method can further comprise: (5) Identifying thereleased identifier oligonucleotide by sequencing the hybridized productproduced in step (4), thereby spatially detecting the at least onetarget analyte in the at least one cell in a tissue sample.

A hybridized product produced in step (4) can be sequenced using anenzyme free method of sequencing. Enzyme-free methods of sequencing havebeen described in, e.g., US2014946386 and U.S. Ser. No. 15/819,151, eachof which is incorporated herein by reference in its entirety.

FIG. 13 shows a schematic of a preferred aspect of a multiplexed enzymefree method of the present disclosure. In this aspect, the probecomprises a target binding domain comprising a nucleic acid sequencethat is complementary to a target nucleic acid. In the top panel, theprobe hybridizes to the target nucleic acid. In the middle panel, a UVphoto-cleavable linker located between the target binding domain and theidentifier oligonucleotide is cleaved, releasing the identifieroligonucleotide. The identifier oligonucleotide comprises a uniquenucleic acid sequence which identifies the target analyte bound to thetarget binding domain, a capture probe binding site, and a multiplexingprobe binding site, as shown in the middle panel. After release, theidentifier oligonucleotide is hybridized to a capture probe and amultiplexing probe as shown in the lower panel. The capture probecomprises a nucleic acid sequence complementary to the capture probebinding site and an affinity molecule. The multiplexing probe comprisesa nucleic acid sequence complementary to the multiplexing probe bindingsite and a nucleic acid sequence which identifies the specific locationof a sample from which the identifier oligonucleotide was released. Thehybridized product is then sequenced using enzyme free sequencingmethods to identify the target nucleic acid bound by the probe.

In one aspect, the present disclosure provides a composition of ahybridized identifier oligonucleotide-capture probe-multiplex probecomplex for spatially detecting at least one target analyte in a sample.A hybridized identifier oligonucleotide-capture probe-multiplex probecomplex comprises an identifier oligonucleotide hybridized to a captureprobe and a multiplexing probe. The identifier oligonucleotide comprisesa unique nucleic acid sequence capable of identifying a specific atarget analyte in a sample, a capture probe binding site and amultiplexing probe binding site. The capture probe comprises an affinitymolecule and a region complementary to the capture probe binding site.The multiplexing probe comprises a nucleic acid sequence whichidentifies the specific location of a sample from which the identifieroligonucleotide was released and a region complementary to themultiplexing probe binding site. A schematic of a hybridized identifieroligonucleotide-capture probe-multiplex probe complex is depicted in thebottom panel of FIG. 13.

FIG. 20 is an exemplary schematic of overview of the methods of thepresent disclosure. First, a sample on a microscope slide is contactedwith a plurality of probes of the present disclosure (step 1 in FIG.20). The slide is then imaged and particular regions of interest (ROIs)are selected (step 2 in FIG. 20). A specific ROI is then illuminated byUV light to release identifier oligonucleotides from probes bound withinthe ROI. The released identifier oligonucleotides are then collected viaaspiration with a microcapillary. Following aspiration, the identifieroligonucleotides are transferred to a particular well within a 96 wellplate. Steps 4 and 5 are then repeated for each ROI identified in step2. After all ROIs have been illuminated and all released identifieroligonucleotides collected, the identifier oligonucleotides aresequenced using next generation sequencing methods to spatially detectat least one target analyte in the sample.

As described in the preceding, the present disclosure provides probesfor the compositions and methods of spatially detecting at least onetarget analyte in a sample. The present disclosure provides probescomprising a target binding domain and an identifier oligonucleotide.The target binding domain is a region of the probe that specificallybinds to at least one target analyte in a sample.

Probes of the present disclosure can be used for spatially detecting atarget nucleic acid. In this aspect, the target binding domain can be atarget nucleic acid-binding region. The target nucleic acid-bindingregion is preferably at least 15 nucleotides in length, and morepreferably is at least 20 nucleotides in length. In specific aspects,the target nucleic acid-binding region is approximately 10 to 500, 20 to400, 25, 30 to 300, 35, 40 to 200, or 50 to 100 nucleotides in length.Probes and methods for binding and identifying a target nucleic acidhave been described in, e.g., US2003/0013091, US2007/0166708,US2010/0015607, US2010/0261026, US2010/0262374, US2010/0112710,US2010/0047924, and US2014/0371088, each of which is incorporated hereinby reference in its entirety.

The target nucleic acid-binding region can directly hybridize to atarget nucleic acid present in a sample. Alternatively, the probes ofthe present disclosure can indirectly hybridize to a target nucleic acidpresent in a sample (via an intermediary oligonucleotide). FIG. 14illustrates a probe (or composition) of this aspect. The probe includesa target nucleic-acid binding domain which binds to a syntheticoligonucleotide (the intermediary oligonucleotide) that in turn binds toa target nucleic acid in a biological sample. It could be said that theintermediary oligonucleotide is a probe, as defined herein, since itcomprises a nucleic acid backbone and is capable of binding a targetnucleic acid. In these aspects, a probe's target nucleic acid-bindingregion hybridizes to a region of an intermediary oligonucleotide (i.e.,a synthetic oligonucleotide) which is different from the target nucleicacid present in a sample. Thus, the probe's target binding region isindependent of the ultimate target nucleic acid in the sample. Thisallows economical and rapid flexibility in an assay design, as thetarget (present in a sample)-specific components of the assay areincluded in inexpensive and widely-available synthetic DNAoligonucleotides rather than the more expensive probes. Such syntheticoligonucleotides are simply designed by including a region thathybridizes to the target nucleic acid present in a sample and a regionthat hybridizes to a probe. Therefore, a single set ofindirectly-binding probes can be used to detect an infinite variety oftarget nucleic acids (present in a sample) in different experimentssimply by replacing the target-specific (synthetic) oligonucleotideportion of the assay.

A target nucleic acid may be DNA or RNA and preferably messenger RNA(mRNA) or miRNA.

Probes of the present disclosure can be used for detecting a targetprotein. In this aspect, the target binding domain can be a targetprotein-binding region. A target protein-binding region includesmolecules or assembles that are designed to bind to at least one targetprotein, at least one target protein surrogate, or both and can, underappropriate conditions, form a molecular complex comprising the probeand the target protein. The target-protein binding region can include anantibody, a peptide, an aptamer, or a peptoid. The antibody can beobtained from a variety of sources, including but not limited topolyclonal antibody, monoclonal antibody, monospecific antibody,recombinantly expressed antibody, humanized antibody, plantibodies, andthe like. The terms protein, polypeptide, peptide, and amino acidsequence are used interchangeably herein to refer to polymers of aminoacids of any length. The polymer may be linear or branched, it maycomprise modified amino acids, and it may be interrupted by non-aminoacids or synthetic amino acids. The terms also encompass an amino acidpolymer that has been modified, for example, by disulfide bondformation, glycosylation, lipidation, acetylation, phosphorylation, orany other manipulation, such as conjugation with a labeling component.As used herein the term amino acid refers to either natural and/orunnatural or synthetic amino acids, including but not limited to glycineand both the D or L optical isomers, and amino acid analogs andpeptidomimetics. Probes and methods for binding and identifying a targetprotein have been described, e.g., in US2011/0086774, the contents ofwhich is incorporated herein by reference in its entirety.

An identifier oligonucleotide is a nucleic acid molecule that identifiesthe target analyte bound to the target binding domain. The identifieroligonucleotide comprises a unique nucleic acid sequence that identifiesthe target analyte bound to the target binding domain of the probe. In anon-limiting example, a probe with a target binding domain that binds tothe protein P53 comprises an identifier oligonucleotide with a uniquenucleic acid sequence that corresponds to P53, while a probe with atarget binding domain that binds to the protein P97 comprises anidentifier oligonucleotide with a unique nucleic acid sequence thatcorresponds to P97.

An identifier oligonucleotide can be DNA, RNA, or a combination of DNAand RNA.

In some aspects, an identifier oligonucleotide can comprise at least oneamplification primer binding site. An amplification primer binding siteis a nucleic acid sequence capable of binding to an amplificationprimer. An amplification primer can be used to amplify the nucleicmolecule to which it is bound using methods known in the art, including,but not limited to, polymerase chain reaction (PCR).

In some aspects, an identifier oligonucleotide can comprise at least oneunique molecular identifier.

An identifier oligonucleotide can be a single-stranded, adouble-stranded, or a partially double-stranded nucleic acid molecule.In the aspects in which an identifier oligonucleotide is double-strandedor partially double-stranded, at least one of the two strands cancomprise at least two separate nucleic acid molecules which, withoutbeing bound by theory, allows for denaturing of the identifieroligonucleotide at lower temperatures.

An identifier oligonucleotide can also comprise at least one 3′ end thatcomprises a single nucleotide overhang.

An identifier oligonucleotide can also comprise a capture probe bindingsite. A capture probe binding site is a nucleic acid sequence to which acapture probe can bind.

A capture probe of the present disclosure can comprise a nucleic acidsequence complementary to a capture probe binding site. A capture probecan also comprise an affinity molecule.

An identifier oligonucleotide can also comprise a multiplexing probebinding site. A multiplexing probe binding site is a nucleic acidsequence to which a multiplexing probe can bind.

A multiplexing probe of the present disclosure can comprise a nucleicacid sequence complementary to a multiplexing probe binding site. Amultiplexing probe can also comprise a nucleic acid sequence whichidentifies the specific location of the tissue sample from which anidentifier oligonucleotide was released.

A probe of the present disclosure can include a region which permits therelease of an identifier oligonucleotide following the application of asuitable force. In one non-limited example, the region is a cleavablemotif (e.g., a restriction enzyme site or cleavable linker). Thecleavable motif allows release of an identifier oligonucleotide from abound target nucleic acid or protein and the identifier oligonucleotidecan then be collected and detected. The region which permits the releaseof an identifier oligonucleotide can be positioned between thetarget-binding domain and the identifier oligonucleotide, allowing forthe release of the identifier oligonucleotide from the target bindingdomain. An identifier oligonucleotide is said to be releasable when itcan be separated (i.e., cleaved and released) from the remainder of theprobe. Examples of cleavable motives include but are not limited tophoto-cleavable linkers. Photo-cleavable linkers can be cleaved by lightprovided by a suitable coherent light source (e.g., a laser and a UVlight source) or a suitable incoherent light source (e.g., an arc-lampand a light-emitting diode (LED)).

In some aspects, the identifier oligonucleotide is collected from asolution proximal to, e.g., at least immediately above or surrounding,the point at which the identifier oligonucleotide is released or the atleast one cell. The proximal solution may be collected by aspirating,e.g., via a pipette, a capillary tube, a microarray pin, a flow cellcomprising holes, or another suitable aspirating system known in the artor any combination thereof. The capillary tube may comprise an opticaldevice capable of transmitting a light force, e.g., UV light, to the atleast one cell. The pipette or a microarray pin may be attached to anarray comprising a plurality of pipettes or microarray pins. Theproximal solution may comprise an anionic polymer, e.g., dextransulfate, and/or salmon sperm DNA and/or the collected signaloligonucleotide may be added to a solution comprising an anionicpolymer, e.g., dextran sulfate, and/or salmon sperm DNA. Othernon-specific blocking agents known in the art in addition to or insteadof salmon sperm DNA may be used.

In some aspects, the identifier oligonucleotide is collected from atissue, at least one cell or proximal to the point at which theidentifier oligonucleotide is released via liquid laminar, turbulent, ortransitional flow. The flow may be via a channel, e.g., having 25 to 500m depth between the tissue and a fluidic device or impermeable barrierplaced over the tissue.

In aspects where the target-binding domain of a probe is an antibody,the probe can be prepared using a cysteine bioconjugation method that isstable, site-specific to, preferably, the antibody's hinge-regionheavy-chain. This preparation method provides relatively controllableidentifier oligonucleotides to antibody stoichiometric ratios. A probecan comprise a plurality (i.e., more than one, e.g., 2, 3, 4, 5, ormore) identifier oligonucleotides per antibody. Generally, “heavier”probes, which comprise 3 or 4 identifier oligonucleotides per antibody,are significantly less sensitive than antibodies lacking an identifieroligonucleotide or “lighter” probes, which comprise 1 or 2 identifieroligonucleotides per antibody.

In aspects, probes are provided to a sample at concentrations typicallyless than that used for immunohistochemistry (IHC) or for in situhybridization (ISH). Alternately, the concentration may be significantlyless than that used for IHC or ISH. For example, the probe concentrationmay be 2 fold less, 5 fold less, 10 fold less, 20 fold less, 25 foldless, 30 fold less, 50 fold less, 60 fold less, 70 fold less, 80 foldless, 90 fold less, 100 fold less, 200 fold less, 300 fold less, 400fold less, 500 fold less, 600 fold less, 700 fold less, 800 fold less,900 fold less, 1000 fold less, 2000 fold less, or less and any number inbetween. In aspects, probes are provided at a concentration of 100 nM,70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM,5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM,0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, and less and any concentrationin between.

Background noise, during protein detection, can be reduced by performinga negative purification of the intact probe molecule. This can be doneby conducting an affinity purification of the antibody orphoto-cleavable linker after collection of eluate from a region ofinterest. Normally, released signal oligonucleotides will not be pulledout of solution. A protein-G or -O mechanism in a pipet tip, tube, orplate can be employed for this step. Such devices and reagentscommercially available.

Background noise, during nucleic acid detection, can be reduced byperforming a negative purification of the intact probe molecule. Thiscan be done by conducting an affinity purification of the target bindingdomain or photo-cleavable linker after collection of eluate from aregion of interest. Normally, released signal oligonucleotides will notbe pulled out of solution. To assist in the negative purification, auniversal purification sequence may be included in a probe, e.g., in thetarget binding domain.

Protein-targeting probes and nucleic acid-targeting probes may beapplied simultaneously as long as conditions allow for binding of both aprotein target and a nucleic acid target. Alternately, protein-targetingprobes and nucleic acid-targeting probes may be applied sequentiallywhen conditions allowing for binding of both a protein target and anucleic acid target are not possible.

A set of probes is synonymous with a composition of probes. A set ofprobes includes at least one species of probes, i.e., directed to onetarget. A set of probes preferably includes at least two, e.g., 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 30, 40, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or morespecies of probes. A probe set may include one or multiple copies ofeach species of probe.

A first set of probes only may be applied to a sample. Alternately, asecond set (or higher number) of probes may be later applied to thesample. The first set and second (or higher number) may target onlynucleic acids, only proteins, or a combination thereof.

In the present disclosure, two or more targets (i.e., proteins, nucleicacids, or a combination thereof) are detected; 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 20, 30, 40, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, 1000 or more targets, and anynumber there between, are detected.

A set of probes may be pre-defined based upon the cell type or tissuetype to be targeted. For example, if the tissue is a breast cancer, thenthe set of probes will include probes directed to proteins relevant tobreast cancer cells (e.g., Her2, EGFR, and PR) and/or probes directed toproteins relevant to normal breast tissues. Additionally, the set ofprobes may be pre-defined based upon developmental status of a cell ortissue to be targeted. Alternately, the set of probes may be pre-definedbased upon subcellular localizations of interest, e.g., nucleus,cytoplasm, and membrane. For example, antibodies directed to Foxp3,Histone H3, or P-S6 label the nucleus, antibodies directed to CD3, CD4,PD-1, or CD45RO label the cytoplasm, and antibodies directed to PD-L1label membranes.

A probe may be chemically synthesized or may be produced biologicallyusing a vector into which a nucleic acid encoding the probe has beencloned.

Any probe or set of probes described herein may be used in methods andkits of the present disclosure.

For the herein-described probes, association of a unique nucleic acidsequence to a specific target nucleic acid or target protein is notfixed.

As described in the preceding, probes of the present disclosure can beused to detect a target nucleic acid or target protein present in anysample, e.g., a biological sample. As will be appreciated by those inthe art, the sample may comprise any number of things, including, butnot limited to: cells (including both primary cells and cultured celllines) and tissues (including cultured or explanted). In aspects, atissue sample (fixed or unfixed) is embedded, serially sectioned, andimmobilized onto a microscope slide. As is well known, a pair of serialsections will include at least one cell that is present in both serialsections. Structures and cell types, located on a first serial sectionwill have a similar location on an adjacent serial section. The samplecan be cultured cells or dissociated cells (fixed or unfixed) that havebeen immobilized onto a slide. A sample can be a formalin-fixedparaffin-embedded (FFPE) tissue sample.

In aspects, a tissue sample is a biopsied tumor or a portion thereof,i.e., a clinically-relevant tissue sample. For example, the tumor may befrom a breast cancer. The sample may be an excised lymph node.

The sample can be obtained from virtually any organism includingmulticellular organisms, e.g., of the plant, fungus, and animalkingdoms; preferably, the sample is obtained from an animal, e.g., amammal. Human samples are particularly preferred.

In some aspects, the probes, compositions, methods, and kits describedherein are used in the diagnosis of a condition. As used herein the termdiagnose or diagnosis of a condition includes predicting or diagnosingthe condition, determining predisposition to the condition, monitoringtreatment of the condition, diagnosing a therapeutic response of thedisease, and prognosis of the condition, condition progression, andresponse to particular treatment of the condition. For example, a tissuesample can be assayed according to any of the probes, methods, or kitsdescribed herein to determine the presence and/or quantity of markers ofa disease or malignant cell type in the sample (relative to thenon-diseased condition), thereby diagnosing or staging a disease or acancer.

In general, samples attached to a slide can be first imaged usingfluorescence (e.g., fluorescent antibodies or fluorescent stains (e.g.,DAPI)) to identify morphology, regions of interest, cell types ofinterest, and single cells and then expression of proteins and/ornucleic acids can be digitally counted from the sample on the sameslide.

Compositions and kits of the present disclosure can include probes andother reagents, for example, buffers and other reagents known in the artto facilitate binding of a protein and/or a nucleic acid in a sample,i.e., for performing hybridization reactions.

A kit also will include instructions for using the components of thekit, including, but not limited to, information necessary to hybridizelabeled oligonucleotides to a probe, to hybridize a probe to atarget-specific oligonucleotide, to hybridize a target-specificoligonucleotide to a target nucleic acid and/or to hybridize a probe totarget protein.

A region of interest may be a tissue type present in a sample, a celltype, a cell, or a subcellular structure within a cell.

Together, a comparison of the identity and abundance of the targetproteins and/or target nucleic acids present in a first region ofinterest (e.g., tissue type, a cell type (including normal and abnormalcells), and a subcellular structure within a cell) and the identity andabundance of the target proteins and/or target nucleic acids present insecond region of interest or more regions of interest can be made usingthe methods of the present disclosure.

As described in the preceding, the products produced by the methods ofthe present disclosure can be used for nucleic acid amplification. In apreferred aspect, the nucleic acid amplification can be solid-phasenucleic acid amplification. Thus, in further aspects the inventionprovides a method of solid-phase nucleic acid amplification of templatepolynucleotide molecules which comprises: preparing a library oftemplate polynucleotide molecules which have common sequences at their5′ and 3′ ends using the methods of the present disclosure and carryingout a solid-phase nucleic acid amplification reaction wherein saidtemplate polynucleotide molecules are amplified. Compositions andmethods for nucleic acid amplification and sequencing have beendescribed in, e.g., U.S. Pat. No. 9,376,678, which is incorporatedherein by reference in its entirety.

The term “solid-phase amplification” as used herein refers to anynucleic acid amplification reaction carried out on or in associationwith a solid support such that all or a portion of the amplifiedproducts are immobilized on the solid support as they are formed. Inparticular, the term encompasses solid-phase polymerase chain reaction(solid-phase PCR), which is a reaction analogous to standard solutionphase PCR, except that one or both of the forward and reverseamplification primers is/are immobilized on the solid support.

Although the invention encompasses “solid-phase” amplification methodsin which only one amplification primer is immobilized (the other primerusually being present in free solution), it is preferred for the solidsupport to be provided with both the forward and the reverse primersimmobilized. In practice, there will be a “plurality” of identicalforward primers and/or a “plurality” of identical reverse primersimmobilized on the solid support, since the PCR process requires anexcess of primers to sustain amplification. References herein to forwardand reverse primers are to be interpreted accordingly as encompassing a“plurality” of such primers unless the context indicates otherwise.

As will be appreciated by the skilled reader, any given PCR reactionrequires at least one type of forward primer and at least one type ofreverse primer specific for the template to be amplified. However, incertain aspects the forward and reverse primers may comprisetemplate-specific portions of identical sequence, and may have entirelyidentical nucleotide sequence and structure (including anynon-nucleotide modifications). In other words, it is possible to carryout solid-phase amplification using only one type of primer, and suchsingle-primer methods are encompassed within the scope of the invention.Other aspects may use forward and reverse primers which containidentical template-specific sequences but which differ in some otherstructural features. For example one type of primer may contain anon-nucleotide modification which is not present in the other.

In other aspects of the invention the forward and reverse primers maycontain template-specific portions of different sequence.

Amplification primers for solid-phase PCR are preferably immobilized bycovalent attachment to the solid support at or near the 5′ end of theprimer, leaving the template-specific portion of the primer free forannealing to its cognate template and the 3′ hydroxyl group free forprimer extension. Any suitable covalent attachment means known in theart may be used for this purpose. The chosen attachment chemistry willdepend on the nature of the solid support, and any derivatization orfunctionalization applied to it. The primer itself may include a moiety,which may be a non-nucleotide chemical modification, to facilitateattachment. In one particularly preferred aspect the primer may includea sulphur-containing nucleophile, such as phosphorothioate orthiophosphate, at the 5′ end. In the case of solid-supportedpolyacrylamide hydrogels (as described below), this nucleophile willbind to a “C” group present in the hydrogel. The most preferred means ofattaching primers and templates to a solid support is via 5′phosphorothioate attachment to a hydrogel comprised of polymerisedacrylamide and N-(5-bromoacetamidylpentyl)acrylamide (BRAPA).

The terms “cluster” and “colony” are used interchangeably herein torefer to a discrete site on a solid support comprised of a plurality ofidentical immobilized nucleic acid strands and a plurality of identicalimmobilized complementary nucleic acid strands. The term “clusteredarray” refers to an array formed from such clusters or colonies. In thiscontext the term “array” is not to be understood as requiring an orderedarrangement of clusters.

The invention also encompasses methods of sequencing the amplifiednucleic acids generated by solid-phase amplification. Thus, theinvention provides a method of nucleic acid sequencing comprisingamplifying a library of nucleic acid templates by the methods of thepresent disclosure described above, using solid-phase amplification asdescribed above to amplify this library on a solid support, and carryingout a nucleic acid sequencing reaction to determine the sequence of thewhole or a part of at least one amplified nucleic acid strand producedin the solid-phase amplification reaction.

Sequencing, as referred to herein, can be carried out using any suitable“sequencing-by-synthesis” technique, wherein nucleotides are addedsuccessively to a free 3′ hydroxyl group, resulting in synthesis of apolynucleotide chain in the 5′ to 3′ direction. The nature of thenucleotide added is preferably determined after each nucleotideaddition.

The initiation point for the sequencing reaction may be provided byannealing of a sequencing primer to a product of the whole genome orsolid-phase amplification reaction. In this connection, one or both ofthe adapters added during formation of the template library may includea nucleotide sequence which permits annealing of a sequencing primer toamplified products derived by whole genome or solid-phase amplificationof the template library.

The products of solid-phase amplification reactions wherein both forwardand reverse amplification primers are covalently immobilized on thesolid surface are so-called “bridged” structures formed by annealing ofpairs of immobilized polynucleotide strands and immobilizedcomplementary strands, both strands being attached to the solid supportat the 5′ end. Arrays comprised of such bridged structures provideinefficient templates for nucleic acid sequencing, since hybridizationof a conventional sequencing primer to one of the immobilized strands isnot favored compared to annealing of this strand to its immobilizedcomplementary strand under standard conditions for hybridization.

In order to provide more suitable templates for nucleic acid sequencingit is preferred to remove substantially all or at least a portion of oneof the immobilized strands in the “bridged” structure in order togenerate a template which is at least partially single-stranded. Theportion of the template which is single-stranded will thus be availablefor hybridization to a sequencing primer. The process of removing all ora portion of one immobilized strand in a “bridged” double-strandednucleic acid structure may be referred to herein as “linearization”.

Bridged template structures may be linearized by cleavage of one or bothstrands with a restriction endonuclease or by cleavage of one strandwith a nicking endonuclease. Other methods of cleavage can be used as analternative to restriction enzymes or nicking enzymes, including interalia chemical cleavage (e.g. cleavage of a diol linkage with periodate),cleavage of abasic sites by cleavage with endonuclease, or by exposureto heat or alkali, cleavage of ribonucleotides incorporated intoamplification products otherwise comprised of deoxyribonucleotides,photochemical cleavage or cleavage of a peptide linker.

It will be appreciated that a linearization step may not be essential ifthe solid-phase amplification reaction is performed with only one primercovalently immobilized and the other in free solution.

In order to generate a linearized template suitable for sequencing it isnecessary to remove “unequal” amounts of the complementary strands inthe bridged structure formed by amplification so as to leave behind alinearized template for sequencing which is fully or partially singlestranded. Most preferably one strand of the bridged structure issubstantially or completely removed.

Following the cleavage step, regardless of the method used for cleavage,the product of the cleavage reaction may be subjected to denaturingconditions in order to remove the portion(s) of the cleaved strand(s)that are not attached to the solid support. Suitable denaturingconditions will be apparent to the skilled reader with reference tostandard molecular biology protocols.

Denaturation (and subsequent re-annealing of the cleaved strands)results in the production of a sequencing template which is partially orsubstantially single-stranded. A sequencing reaction may then beinitiated by hybridization of a sequencing primer to the single-strandedportion of the template.

Thus, the nucleic acid sequencing reaction may comprise hybridizing asequencing primer to a single-stranded region of a linearizedamplification product, sequentially incorporating one or morenucleotides into a polynucleotide strand complementary to the region ofamplified template strand to be sequenced, identifying the base presentin one or more of the incorporated nucleotide(s) and thereby determiningthe sequence of a region of the template strand.

One preferred sequencing method which can be used in accordance with theinvention relies on the use of modified nucleotides that can act aschain terminators. Once the modified nucleotide has been incorporatedinto the growing polynucleotide chain complementary to the region of thetemplate being sequenced there is no free 3′-OH group available todirect further sequence extension and therefore the polymerase cannotadd further nucleotides. Once the nature of the base incorporated intothe growing chain has been determined, the 3′ block may be removed toallow addition of the next successive nucleotide. By ordering theproducts derived using these modified nucleotides it is possible todeduce the DNA sequence of the DNA template. Such reactions can be donein a single experiment if each of the modified nucleotides has attacheda different label, known to correspond to the particular base, tofacilitate discrimination between the bases added at each incorporationstep. Alternatively, a separate reaction may be carried out containingeach of the modified nucleotides separately.

The modified nucleotides may carry a label to facilitate theirdetection. Preferably this is a fluorescent label. Each nucleotide typemay carry a different fluorescent label. However the detectable labelneed not be a fluorescent label. Any label can be used which allows thedetection of an incorporated nucleotide.

One method for detecting fluorescently labelled nucleotides comprisesusing laser light of a wavelength specific for the labelled nucleotides,or the use of other suitable sources of illumination. The fluorescencefrom the label on the nucleotide may be detected by a CCD camera orother suitable detection means.

The invention is not intended to be limited to use of the sequencingmethod outlined above, as essentially any sequencing methodology whichrelies on successive incorporation of nucleotides into a polynucleotidechain can be used. Suitable alternative techniques include, for example,Pyrosequencing, FISSEQ (fluorescent in situ sequencing), MPSS (massivelyparallel signature sequencing) and sequencing by ligation-based methods.

In methods of the present disclosure, the unique nucleic acid sequencepresent in the identifier oligonucleotide of a probe which identifiesthe target analyte bound to the target binding domain of the probe cancomprise between about 5 nucleotides and about 50 nucleotides.Preferably, the sequence comprises between about 20 nucleotides andabout 40 nucleotides. Even more preferably, the sequence comprises about35 nucleotides. In some preferred aspects, the sequence comprises 10nucleotides.

In methods of the present disclosure, the nucleic acid sequence whichidentifies the specific location of the tissue sample from which theidentifier oligonucleotide was released comprises between about 6nucleotides and about 15 nucleotides. Preferably, the sequence comprisesabout 12 nucleotides.

In methods of the present disclosure, an amplification primer bindingsites comprises between about 18 nucleotides and about 40 nucleotides.Preferably, an amplification primer binding sites comprises about 32nucleotides.

In some aspects of the methods of the present disclosure, anamplification primer binding site can comprise an i7 sequence, whereinthe i7 sequence comprises the sequence set forth in SEQ ID NO: 1.

In some aspects of the methods of the present disclosure, anamplification primer binding site can comprise an i5 sequence, whereinthe i5 sequence comprises the sequence set forth in SEQ ID NO: 2.

In some aspects of the methods of the present disclosure, anamplification primer can comprise a flow cell adapter sequence, whereinthe flow cell adapter sequence is suitable for sequencing. Preferably,at least one amplification primer used in the methods of the presentdisclosure comprises a P5 flow cell adapter sequence, wherein the P5flow cell adapter sequence comprises the sequence set forth in SEQ IDNO: 3. Preferably still, at least one amplification primer used in themethods of the present disclosure comprises a P7 flow cell adaptersequence, wherein the P7 flow cell adapter sequence comprises thesequence set forth in SEQ ID NO: 4.

In methods of the present disclosure, a unique molecular identifier cancomprise between about 6 nucleotides and about 30 nucleotides.Preferably, a unique molecular identifier can comprise about 15nucleotides. The terms unique molecular identifier and random moleculartags are used interchangeably herein. Using methods known in that art,unique molecular identifiers are random sequences that can be used tocorrect for biases in amplification prior to sequencing.

In methods of the present disclosure, a constant nucleic acid sequenceto minimize ligation bias comprises between about 1 nucleotide and about15 nucleotides. Preferably, the constant sequence comprises about 8nucleotides.

In some aspects, a flow cell binding site can comprise between about 15to about 40 nucleotides. A flow cell binding site can comprise about 29nucleotides. A flow cell binding site can comprise about 24 nucleotides.

In some aspects, a target binding domain can comprise between about 10to about 70 nucleotides. A target binding domain can comprise betweenabout 30 to about 55 nucleotides. A target binding domain can comprisebetween about 35 to about 50 nucleotides.

In some aspects, a unique nucleic acid sequence which identifies thetarget analyte bound to the target binding domain can comprise betweenabout 20 to about 40. A unique nucleic acid sequence which identifiesthe target analyte bound to the target binding domain can comprise about25 nucleotides, or about 35 nucleotides, or about 12 nucleotides.

In some aspects, an amplification primer binding site can comprisebetween about 20 to about 50 nucleotides. An amplification primerbinding site can comprise about 33 nucleotides, or about 34 nucleotides.

In some aspects, a nucleic acid sequence which identifies the specificlocation of the tissue sample from which the identifier oligonucleotidewas released can comprise between about 1 to about 20 nucleotides. Anucleic acid sequence which identifies the specific location of thetissue sample from which the identifier oligonucleotide was released cancomprise about 8 nucleotides.

In some aspects, a nucleic acid sequence comprising a unique molecularidentifier can comprise between about 5 to about 20 nucleotides. Anucleic acid sequence comprising a unique molecular identifier cancomprise about 14 nucleotides.

As used herein, the terms “region of interest” and “ROI” are used intheir broadest sense to refer to a specific location within a samplethat is to be analyzed using the methods of the present disclosure.

As used herein, the term “adjacent” can mean within about 1 nucleotide,or within about 2 nucleotides, or within about 3 nucleotides, or withinabout 4 nucleotides, or within about 5 nucleotides, or within about 6nucleotides, or within about 7 nucleotides, or within about 8nucleotides, or within about 9 nucleotides, or within about 10nucleotides, or within about 11 nucleotides, or within about 12nucleotides, or within about 13 nucleotides, or within about 14nucleotides, or within about 15 nucleotides, or within about 16nucleotides, or within about 17 nucleotides, or within about 18nucleotides, or within about 19 nucleotides, or within about 20nucleotides, or within about 21 nucleotides, or within about 22nucleotides, or within about 23 nucleotides, or within about 24nucleotides, or within about 25 nucleotides, or within about 26nucleotides, or within about 27 nucleotides, or within about 28nucleotides, or within about 29 nucleotides, or within about 30nucleotides, or within about 40 nucleotides, or within aboutnucleotides, or within about 50 nucleotides, or within about 60nucleotides, or within about 70 nucleotides, or within about 80nucleotides, or within about 90 nucleotides, or within about 100nucleotides.

As used herein, the term “spatially detecting” is used in its broadestsense to refer to the identification of the presence of a specifictarget analyte within a specific region of interest in a sample.Spatially detecting can comprise quantifying the amount of a specifictarget analyte present within a specific region of interest in a sample.Spatially detecting can further comprise quantifying the relative amountof a first target analyte within a specific region of interest in asample as compared to the amount of at least a second target analytewithin a specific region of interest in a sample. Spatially detectingcan also comprise quantifying the relative amount of a specific targetanalyte within a first region of interest in a sample compared to theamount of the same target analyte in at least a second region ofinterest in the same sample or different sample.

In some aspects of the methods and compositions of the presentdisclosure, a target analyte can be any molecule within a sample that isto be spatially detected. Target analytes include, but are not limitedto, nucleic acid molecules and protein molecules. When the targetanalyte is a protein, the protein can be referred to as a targetprotein. When the target analyte is a nucleic acid, the nucleic acid canbe referred to as a target nucleic acid. Target nucleic acids caninclude, but are not limited to, mRNA molecules, micro RNA (miRNA)molecules, tRNA molecules, rRNA molecules, gDNA or any other nucleicacid present within a sample.

In some aspects of the methods and compositions of the presentdisclosure, the term target binding domain is used in its broadest senseto refer to a portion of a probe of the present disclosure that bindsto, either directly or indirectly, a target analyte located in a sample.A target binding domain can comprise nucleic acid, protein, at least oneantibody, an aptamer, or any combination thereof. A target bindingdomain can comprise DNA, RNA or any combination thereof. A targetbinding domain can comprise any number of modified nucleotides and/ornucleic acid analogues.

In the aspect that the target analyte to be spatially detected is atarget protein, a target binding domain can be a protein-target bindingdomain. A protein-target binding domain can comprise an antibody orantibody fragment that binds to the target protein.

In the aspect that the target analyte to be spatially detected is atarget nucleic acid, a target binding domain can be a target nucleicacid-binding region. A target nucleic acid-binding region can comprise anucleic acid that is complementary to the target nucleic acid to bespatially detected. A target nucleic acid-binding region can comprise anucleic acid that hybridizes to the target nucleic acid to be detected.

As used herein, the term “hybridize” is used in its broadest sense tomean the formation of a stable nucleic acid duplex. In one aspect,“stable duplex” means that a duplex structure is not destroyed by astringent wash under conditions such as, for example, a temperature ofeither about 5° C. below or about 5° C. above the Tm of a strand of theduplex and low monovalent salt concentration, e.g., less than 0.2 M, orless than 0.1 M or salt concentrations known to those of skill in theart. A duplex can be “perfectly matched”, such that the polynucleotideand/or oligonucleotide strands making up the duplex form a doublestranded structure with one another such that every nucleotide in eachstrand undergoes Watson-Crick base pairing with a nucleotide in theother strand. The term “duplex” comprises, but is not limited to, thepairing of nucleoside analogs, such as deoxyinosine, nucleosides with2-aminopurine bases, PNAs, and the like, that can be employed. A duplexcan comprise at least one mismatch, wherein the term “mismatch” meansthat a pair of nucleotides in the duplex fail to undergo Watson-Crickbonding.

As used herein, the term “hybridization conditions,” will typicallyinclude salt concentrations of less than about 1 M, more usually lessthan about 500 mM and even more usually less than about 200 mM.Hybridization temperatures can be as low as 5° C., but are typicallygreater than 22° C., more typically greater than about 30° C., and oftenin excess of about 37° C. Hybridizations are usually performed understringent conditions, e.g., conditions under which a probe willspecifically hybridize to its target analyte. Stringent conditions aresequence-dependent and are different in different circumstances. Longerfragments can require higher hybridization temperatures for specifichybridization. As other factors can affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, the combination of parameters is more important than theabsolute measure of any one alone. Certain hybridization conditions willpromote the formation of a duplex between the entire length of a targetbinding domain and the target analyte. Other hybridization conditionswill promote the formation of a duplex only along certain portions ofthe target binding domain.

In some aspects of the methods and compositions of the presentdisclosure, a probe can comprise a target binding domain directly, orindirectly linked to an identifier oligonucleotide. In the context of aprobe, an identifier oligonucleotide is a polynucleotide that comprisesa nucleic acid sequence that identifies the target analyte bound to thetarget binding domain of that probe. That is to say, the identifieroligonucleotide comprises a specific nucleic acid sequence that is apriori assigned to the specific target analyte bound to the targetbinding to which the identifier oligonucleotide is attached. In anon-limiting example, a probe designated as “probe X” designed tospatially detect “target analyte X” comprises a target binding domaindesignated “target binding domain X” linked to an identifieroligonucleotide designated “identifier oligonucleotide X”. Targetbinding domain X binds to target analyte X and identifieroligonucleotide X comprises a nucleic acid sequence, designated as“nucleic acid sequence X”, which corresponds to target analyte X Thus,if a skilled artisan practicing the methods of the present disclosurewere to collect identifier oligonucleotides released from a region ofinterest in sample and obtain nucleic acid sequence X after sequencing,the skilled artisan would understand that to indicate that targetanalyte X was present in that region of interest. The amount, or numberof sequencing reads, of nucleic acid sequence X can be used to determinethe quantify, in absolute or relative terms, the amount of targetanalyte X within the region of interest.

As used herein, the term “amplification primer binding site” is used inits broadest sense to refer to a nucleic acid sequence that iscomplementary to, or at least partially complementary to at least oneamplification primer, wherein the amplification primer is a shortsingle-stranded or partially single-stranded oligonucleotide that issufficient to prime DNA and/or RNA synthesis, for example, by PCR.

In some aspects of the methods and compositions of the presentdisclosure, a target binding domain can be linked to an identifieroligonucleotide by a cleavable linker. Suitable cleavable linkersinclude, but are not limited to, chemically cleavable linkers (e.g. alinker that is cleaved when exposed to a particular chemical,combination of chemicals or reaction conditions), a photo-cleavablelinker (e.g. a linker that is cleaved when exposed to light of asufficient wavelength or light comprising a sufficient range ofwavelengths), or an enzymatically cleavable linker (e.g. a linker thatis cleaved by a specific enzyme or class of enzymes). Thus, as usedherein the phrase “providing a force to a location of the samplesufficient to release an identifier oligonucleotide” is used in itsbroadest sense to describe changing the conditions within a certainregion of interest in a sample such that, for any probe bound to atarget analyte within that region of interest, the linker between thetarget binding domain of the probe and the identifier oligonucleotide ofthe probe is cleaved, thereby separating the identifier oligonucleotidefrom the target binding domain so that the identifier oligonucleotidecan be subsequently collected from solution. For example, in aspectswherein a probe comprises a chemically cleavable linker between thetarget binding domain and the identifier oligonucleotide, providing aforce to a location of the sample sufficient to release an identifieroligonucleotide can comprise exposing that location of the sample to thespecific chemical, combination of chemicals or reaction conditions thatcatalyze the cleavage of the linker. In another non-limiting example, inaspects wherein a probe comprises a photo-cleavable linker between thetarget binding domain and the identifier oligonucleotide, providing aforce to a location of the sample sufficient to release an identifieroligonucleotide can comprise exposing/exciting that location of thesample with light of a sufficient wavelength capable of cleaving thephoto-cleavable linker. In another non-limiting example, in aspectswherein a probe comprises an enzymatically cleavable linker between thetarget binding domain and the identifier oligonucleotide, providing aforce to a location of the sample sufficient to release an identifieroligonucleotide can comprise exposing that location of the sample to anamount of enzyme sufficient to catalyze the cleavage of the linker.

Providing a force to a location of the sample sufficient to release anidentifier oligonucleotide can result in at least about 10%, or at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 85%, or at least about 90%, or at leastabout 95%, or at least about 99% of probes bound to target analyteswithin that location of the sample to undergo cleavage of the linkerconnecting the target binding domain and the identifier oligonucleotide.

As would be appreciated by one skilled in the art, the term “uniquemolecular identifier” or “UMI” refer to short nucleic acid sequencesthat are used to quantify and reduce quantitative bias caused by nucleicacid amplification prior to sequencing reactions.

In some aspects of the methods and compositions of the presentdisclosure, an affinity moiety can comprise biotin, avidin,streptavidin, nucleic acid, or any combination thereof.

In some aspects of the methods and compositions of the presentdisclosure, a probe can comprise at least at least about 5, about 10, atleast about 15, at least about 20, at least about 25, at least about 30,at least about 35 at least about 40, at least about 45, at least about50, at least about 55, at least about 60, at least about 65, at leastabout 70, at least about 75, at least about 80, at least about 85, atleast about 90, at least about 95, at least about 100, at least about110, at least about 120, at least about 130, at least about 140, atleast about 150, at least about 160, at least about 170, at least about180, at least about 190 or at least about 200 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a target binding domain can comprise at least at least about5, about 10, at least about 15, at least about 20, at least about 25, atleast about 30, at least about 35 at least about 40, at least about 45,at least about 50, at least about 55, at least about 60, at least about65, at least about 70, at least about 75, at least about 80, at leastabout 85, at least about 90, at least about 95, at least about 100, atleast about 110, at least about 120, at least about 130, at least about140, at least about 150, at least about 160, at least about 170, atleast about 180, at least about 190 or at least about 200 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, an identifier oligonucleotide can comprise at least about 5,at least about 10, at least about 15, at least about 20, at least about25, at least about 30, at least about 35 at least about 40, at leastabout 45, at least about 50, at least about 55, at least about 60, atleast about 65, at least about 70, at least about 75, at least about 80,at least about 85, at least about 90, at least about 95, at least about100, at least about 110, at least about 120, at least about 130, atleast about 140, at least about 150, at least about 160, at least about170, at least about 180, at least about 190 or at least about 200nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, an amplification primer can comprise at least about 5, atleast about 10, at least about 15, at least about 20, at least about 25,at least about 30, at least about 35 at least about 40, at least about45, at least about 50, at least about 55, at least about 60, at leastabout 65, at least about 70, at least about 75, at least about 80, atleast about 85, at least about 90, at least about 95, at least about100, at least about 110, at least about 120, at least about 130, atleast about 140, at least about 150, at least about 160, at least about170, at least about 180, at least about 190 or at least about 200nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a nucleic acid probe can comprise at least about 5, at leastabout 10, at least about 15, at least about 20, at least about 25, atleast about 30, at least about 35 at least about 40, at least about 45,at least about 50, at least about 55, at least about 60, at least about65, at least about 70, at least about 75, at least about 80, at leastabout 85, at least about 90, at least about 95, at least about 100, atleast about 110, at least about 120, at least about 130, at least about140, at least about 150, at least about 160, at least about 170, atleast about 180, at least about 190 or at least about 200 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a nucleic acid complementary to a portion of a identifieroligonucleotide can comprise at least about 5, at least about 10, atleast about 15, at least about 20, at least about 25, at least about 30,at least about 35 at least about 40, at least about 45, at least about50, at least about 55, at least about 60, at least about 65, at leastabout 70, at least about 75, at least about 80, at least about 85, atleast about 90, at least about 95 or at least about 100 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a nucleic acid sequence comprising a molecular identifiercan comprise at least about 5, or at least about 10 nucleotides, or atleast about 15, or at least about 20, or at least about 25, or at leastabout 30, or at least about 35, or at least about 40, or at least about45, or at least about 50 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, an amplification primer binding site can comprise at leastabout 5, at least about 10, at least about 15, at least about 20, atleast about 25, at least about 30, at least about 35, at least about 40,at least about 45, at least about 50, at least about 55, at least about60, at least about 65 or at least about 70 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a flow cell adapter sequence suitable for sequencing cancomprise at least about 5, at least about 10, at least about 15, atleast about 20, at least about 25, at least about 30, at least about 35at least about 40, at least about 45, at least about 50, at least about55, at least about 60, at least about 65, at least about 70, at leastabout 75, at least about 80, at least about 85, at least about 90, atleast about 95 or at least about 100 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a flow cell binding site can comprise at least about 5, atleast about 10, at least about 15, at least about 20, at least about 25,at least about 30, at least about 35, at least about 40, at least about45, at least about 50, at least about 55, at least about 60, at leastabout 65, at least about 70, at least about 75, at least about 80, atleast about 90, at least about 95 or at least about 100 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a nucleic acid sequence which identifies the specificlocation of the tissue sample from which an identifier oligonucleotidewas released can comprise at least about 5, or at least about 10nucleotides, or at least about 15, or at least about 20, or at leastabout 25, or at least about 30, or at least about 35, or at least about40, or at least about 45, or at least about 50 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a unique nucleic acid sequence which identifies the targetanalyte bound to a target binding domain can comprise at least about 5,at least about 10, at least about 15, at least about 20, at least about25, at least about 30, at least about 35, at least about 40, at leastabout 45, at least about 50, at least about 55, at least about 60, atleast about 65, at least about 70, at least about 75, at least about 80,at least about 90, at least about 95 or at least about 100 nucleotides.

In some aspects of the methods and compositions of the presentdisclosure, a probe, a target binding domain, an identifieroligonucleotide, an amplification primer, a nucleic acid probe, anucleic acid complementary to a portion of a identifier oligonucleotide,a nucleic acid sequence comprising a molecular identifier, anamplification primer binding site, a flow cell adapter sequence, a flowcell binding site, a nucleic acid sequence which identifies the specificlocation of the tissue sample from which an identifier oligonucleotidewas released, a unique nucleic acid sequence which identifies the targetanalyte bound to a target binding domain or any combination thereof cancomprise at least one natural base, can comprise no natural bases, cancomprise at least one modified nucleotide or nucleic acid analog, cancomprise no modified nucleotides or nucleic acid analogs, can compriseat least one universal base, can comprise no universal bases, cancomprise at least one degenerate base or can comprise no degeneratebases.

In some aspects of the methods and compositions of the presentdisclosure, a probe, a target binding domain, an identifieroligonucleotide, an amplification primer, a nucleic acid probe, anucleic acid complementary to a portion of a identifier oligonucleotide,a nucleic acid sequence comprising a molecular identifier, anamplification primer binding site, a flow cell adapter sequence, a flowcell binding site, a nucleic acid sequence which identifies the specificlocation of the tissue sample from which an identifier oligonucleotidewas released, a unique nucleic acid sequence which identifies the targetanalyte bound to a target binding domain or any combination thereof cancomprise any combination natural bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more natural bases), modified nucleotides or nucleic acidanalogs (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified oranalog nucleotides), universal bases (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more universal bases), or degenerate bases (e.g. 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more degenerative bases). When present in acombination, the natural bases, modified nucleotides or nucleic acidanalogs, universal bases and degenerate bases can be arranged in anyorder.

The terms “modified nucleotides” or “nucleic acid analogues” include,but are not limited to, locked nucleic acids (LNA), bridged nucleicacids (BNA), propyne-modified nucleic acids, zip nucleic acids (ZNA®),isoguanine and isocytosine. Preferably, the modified nucleotides ornucleic acid analogues are locked nucleic acids (LNAs).

The term “locked nucleic acids (LNA)” as used herein includes, but isnot limited to, a modified RNA nucleotide in which the ribose moietycomprises a methylene bridge connecting the 2′ oxygen and the 4′ carbon.This methylene bridge locks the ribose in the 3′-endo confirmation, alsoknown as the north confirmation, that is found in A-form RNA duplexes.The term inaccessible RNA can be used interchangeably with LNA. The term“bridged nucleic acids (BNA)” as used herein includes, but is notlimited to, modified RNA molecules that comprise a five-membered orsix-membered bridged structure with a fixed 3′-endo confirmation, alsoknown as the north confirmation. The bridged structure connects the 2′oxygen of the ribose to the 4′ carbon of the ribose. Various differentbridge structures are possible containing carbon, nitrogen, and hydrogenatoms. The term “propyne-modified nucleic acids” as used hereinincludes, but is not limited to, pyrimidines, namely cytosine andthymine/uracil, that comprise a propyne modification at the C5 positionof the nucleic acid base. The term “zip nucleic acids) (ZNA®)” as usedherein includes, but is not limited to, oligonucleotides that areconjugated with cationic spermine moieties.

The term “universal base” as used herein includes, but is not limitedto, a nucleotide base does not follow Watson-Crick base pair rules butrather can bind to any of the four canonical bases (A, T/U, C, G)located on the target nucleic acid. The term “degenerate base” as usedherein includes, but is not limited to, a nucleotide base that does notfollow Watson-Crick base pair rules but rather can bind to at least twoof the four canonical bases A, T/U, C, G), but not all four. Adegenerate base can also be termed a Wobble base; these terms are usedinterchangeably herein.

As used in this Specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive and covers both “or” and “and”.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromthe context, all numerical values provided herein are modified by theterm “about.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although other probes,compositions, methods, and kits similar, or equivalent, to thosedescribed herein can be used in the practice of the present disclosure,the preferred materials and methods are described herein. It is to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only, and is not intended to be limiting.

EXAMPLES Example 1—Two-Ended Adapter Ligation Method for 96 MultiplexedSamples

In this example, a two-ended adapter ligation method of the presentdisclosure was used to sequence identifier oligonucleotides collectedfrom 96 multiplexed samples. The nucleic acid adapters used in thisexperiment were partially double-stranded. The nucleic acid adapterscomprised a first strand and a second strand. The first strand compriseda 5′ phosphate moiety for ligation. The first strand also comprised aconstant nucleic acid sequence to minimize ligation bias (GCGTAGTG), anucleic acid sequence comprising a unique molecular identifier, a uniquenucleic acid sequence which identifies the specific location of thesample from which the identifier oligonucleotide was released and afirst amplification primer binding site (SEQ ID NO: 2). The secondstrand comprised a single overhanging thymine nucleotide at the 3′ end,a sequence complementary to the constant nucleic acid sequence tominimize ligation bias present in the first strand, a sequencecomplementary to the unique nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released present in the first strand and a secondamplification primer binding site (SEQ ID NO: 1).

To form the partially double stranded nucleic acid adapters, firststrand oligonucleotides and second strand oligonucleotides were combinedin equimolar proportion for a final total oligonucleotide concentrationof 28 μM in buffer comprising 50 mM NaCl. The oligonucleotide mixturewas heated at 95° C. for 2 minutes and cooled at ambient temperature for30 minutes, thereby annealing the first stand and second strandoligonucleotides together to form the partially double-stranded nucleicacid adapters. Annealed nucleic acid adaptors were diluted to finalconcentration ranging between 0.02 μM to 0.002 μM in a solution of 10 mMTris pH 8 and 0.05% Tween20.

Collected identifier oligonucleotides were end repaired and A-tailedusing NEBNext Ultra II DNA Library Prep Kit for Illumina (New EnglandBiolabs) with a modified protocol. End repair/A-tail master mix wasprepared by combining the following: 627.8 μL of PCR-grade H2O, 143.9 μLof NEBNext Ultra II End Prep Reaction Buffer, and 61.7 μL of NEBNextUltra II End Prep Enzyme Mix. 8.3 μL of end repair/A-tail master mix wasadded to 4 μL of each sample of identifier oligonucleotides. Thereaction was incubated for 30 minutes at 20° C. with a heated lidof >75° C., followed by a second incubation for 30 minutes at 65° C. Therepaired/A-tailed identifier oligonucleotide mixtures were then storedat 4° C.

Following end repair and A-tailing, the nucleic acid adaptors wereligated to the repaired/A-tailed identifier oligonucleotides by adding6.4 μL of NEBNext Ultra II Ligation Master Mix, 0.2 μL of NEBNextLigation Enhancer, and 1 μL of the nucleic acid adapter dilution to eachrepaired/A-tailed identifier oligonucleotide mixture. These reactionswere incubated for 15 minutes at 20° C. with the heated lid off andsubsequently quenched with 1 μL 0.5M EDTA. All of the reactions werethen pooled into a single 15 mL conical tube to form a pooledadapter-ligated sample.

The pooled adaptor-ligated sample was purified using diluted AgencourtAMPure XP magnetic beads (Beckman Coulter Genomics Inc.), which wereprepared by combining 350 of AMPure XP beads and 3.15 mL of AMPure XPbuffer (2.5M NaCl, 20% PEG8000). AMPure XP bead cleanup was performedwith 3.5 mL of diluted AMPure XP beads and eluted in 200 of a buffercomprising 10 mM Tris pH 8 and 0.05% Tween20. AMPure XP bead cleanup wasthen repeated with 400 μL of AMPure XP beads and eluted in 20 μL of abuffer comprising 10 mM Tris pH 8 and 0.05% Tween20 to obtain purifiedadapter-ligated samples.

Following AMPure XP cleanup, PCR reactions with purified adaptor-ligatedsample were prepared to amplify the adapter-ligated identifieroligonucleotides. To 6 μL of the purified adapter-ligated sample, 10 μLof NEBNext Ultra II Q5 Master Mix, 0.2 μL of 100 μM forward and reverseprimers, and 3.6 μL of PCR-grade H2O was added. The forward primerscomprised a flow cell adapter sequence suitable for sequencing, a uniquemolecular identifier and a nucleic acid sequence complementary to thefirst amplification primer binding site located on the first strand ofthe nucleic acid adapter. Table 1 provides the sequences of the forwardprimers used.

TABLE 1 Forward primers for two-ended adapter ligation SEQ IDPrimer Sequence NO AATGATACGGCGACCACCGAGATCTACACG 5CTCAGATATAGCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 6 CTCAGAATAGAGGCACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 7CTCAGACCTATCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 8 CTCAGAGGCTCTGAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 9CTCAGAAGGCGAAGACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 10 CTCAGATAATCTTAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 11CTCAGACAGGACGTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 12 CTCAGAGTACTGACACACTCTTTAAGACGACGTCGCTATGGCCTCTCC

The reverse primers comprised a flow cell adapter sequence suitable forsequencing, a unique molecular identifier and a nucleic acid sequencecomplementary to the second amplification primer binding site located onthe second strand of the nucleic acid adapter. Table 2 provides thesequences of the reverse primers used.

TABLE 2 Reverse primers for two-ended adapter ligation SEQ IDPrimer Sequence NO CAAGCAGAAGACGGCATACGAGATCGAGTA 13ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCTCCG 14GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAATGAG 15CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGAATC 16TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTTCTGA 17ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATACGAAT 18TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGCTTC 19AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCGCAT 20TAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCATAGC 21CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTTCGCG 22GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCGCGA 23GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTATCG 24CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT

The optimal number of PCR cycles was determined empirically withtriplicate PCR reactions. Alternately, the optimal number of PCR cyclescould have been determined using real-time/qPCR. The PCR program usedcomprised the following steps:

(1) 30 seconds at 98° C.

(2) 10 seconds at 98° C.

(3) 1 minute at 65° C.

(4) Repeating steps (2) and (3) nine times

(5) 5 minutes at 65° C.

The amplified products were purified using 18 μL of AMPure XP beads andeluting with 20 μL of a buffer comprising 10 mM Tris pH 8 and 0.05%Tween20.

The amplified products were assessed using a High Sensitivity DNA chipon 2100 Bioanalyzer (Agilent Genomics) and KAPA Library QuantificationKit for Illumina Platforms (Kapa Biosystems). The amplified productswere also diluted to 15 pM for sequencing on a MiSeq (Illumina)according to manufacturer's instructions (MiSeq Reagent Kit v3 2×75 bp)with a custom spike-in primer comprising the nucleotide sequence

(SEQ ID NO: 25) ACACTCTTTAAGACGACGTCGCTATGGCCTCTCC.

Example 2—One-Ended Adapter Ligation Method for 96 Multiplexed Samples

In this example, a one-ended adapter ligation method of the presentdisclosure was used to sequence identifier oligonucleotides collectedfrom 96 multiplexed samples. The nucleic acid adapters used in thisexperiment were partially double-stranded. The nucleic acid adapterscomprised a first strand and a second strand. The first strand compriseda 5′ phosphate moiety for ligation. The first strand also comprised aconstant nucleic acid sequence to minimize ligation bias (CACTACGC), anucleic acid sequence comprising a unique molecular identifier, a uniquenucleic acid sequence which identifies the specific location of thesample from which the identifier oligonucleotide was released and afirst amplification primer binding site (SEQ ID NO: 1). The secondstrand comprised a single overhanging thymine nucleotide at the 3′ end,a sequence complementary to the constant nucleic acid sequence tominimize ligation bias present in the first strand and a sequencecomplementary to the unique nucleic acid sequence which identifies thespecific location of the sample from which the identifieroligonucleotide was released present in the first strand.

To form the partially double stranded nucleic acid adapters, firststrand oligonucleotides and second strand oligonucleotides were combinedin equimolar proportion for a final total oligonucleotide concentrationof 28 μM in 50 mM NaCl. The oligonucleotide mixture was heated to 95° C.for 2 minutes and cooled at ambient temperature for 30 minutes, therebyannealing the first stand and second strand oligonucleotides together toform the partially double-stranded nucleic acid adapters. Annealednucleic acid adaptors were diluted to a final concentration rangingbetween 0.02 μM to 0.002 μM in a solution of 10 mM Tris pH 8 and 0.05%Tween20.

Nucleic acid adapters were ligated to the collected identifieroligonucleotides by addition of 10 μL of 2× rapid ligation buffer(Enzymatics), 1 μL of T4 DNA Rapid Ligase (Enzymatics), and 1 μL ofannealed nucleic acid adapter dilutions to each sample of collectedidentifier oligonucleotides. Samples were incubated for 15 minutes at20° C. with the heated lid off and subsequently quenched with 1 μL 0.5MEDTA. All of the reactions were then pooled into a single 15 mL conicaltube to form a pooled adapter-ligated sample.

The pooled adaptor-ligated sample was purified using diluted AgencourtAMPure XP magnetic beads (Beckman Coulter Genomics Inc.), which wereprepared by combining 350 of AMPure XP beads and 3.15 mL of AMPure XPbuffer (2.5M NaCl, 20% PEG8000). AMPure XP bead cleanup was performedwith 3.5 mL of diluted AMPure XP beads and eluted in 200 of a buffercomprising 10 mM Tris pH 8 and 0.05% Tween20. AMPure XP bead cleanup wasthen repeated with 400 μL of AMPure XP beads and eluted in 20 μL of abuffer comprising 10 mM Tris pH 8 and 0.05% Tween20 to obtain purifiedadapter-ligated samples.

Following AMPure XP cleanup, PCR reactions with purified adaptor-ligatedsample were prepared to amplify the adapter-ligated identifieroligonucleotides. To 6 μL of the purified adapter-ligated sample, 10 μLof NEBNext Ultra II Q5 Master Mix, 0.2 μL of 100 μM forward and reverseprimers, and 3.6 μL of PCR-grade H₂O was added. The forward primerscomprised a flow cell adapter sequence suitable for sequencing, a uniquemolecular identifier and a nucleic acid sequence complementary to thefirst amplification primer binding site located on the first strand ofthe nucleic acid adapter. Table 3 provides the sequences of the forwardprimers used.

TABLE 3 Forward primers for two-ended adapter ligation SEQ IDPrimer Sequence NO AATGATACGGCGACCACCGAGATCTACACG 5CTCAGATATAGCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 6 CTCAGAATAGAGGCACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 7CTCAGACCTATCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 8 CTCAGAGGCTCTGAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 9CTCAGAAGGCGAAGACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 10 CTCAGATAATCTTAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 11CTCAGACAGGACGTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 12 CTCAGAGTACTGACACACTCTTTAAGACGACGTCGCTATGGCCTCTCC

The reverse primers comprised a flow cell adapter sequence suitable forsequencing, a unique molecular identifier and a nucleic acid sequencecomplementary to the second amplification primer binding site located onthe second strand of the nucleic acid adapter. Table 4 provides thesequences of the reverse primers used.

TABLE 4 Reverse primers for two-ended adapter ligation SEQ IDPrimer Sequence NO CAAGCAGAAGACGGCATACGAGATCGAGTA 13ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCTCCG 14GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAATGAG 15CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGAATC 16TCGTGACTGGAGTTCAGACGTGT GCTCTT CCGATCT CAAGCAGAAGACGGCATACGAGATTTCTGA 17ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATACGAAT 18TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGCTTC 19AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCGCAT 20TAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCATAGC 21CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTTCGCG 22GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCGCGA 23GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTATCG 24CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT

The optimal number of PCR cycles was determined empirically withtriplicate PCR reactions. Alternately, the optimal number of PCR cyclescould have been determined using real-time/qPCR. The PCR program usedcomprised the following steps:

(1) 30 seconds at 98° C.

(2) 10 seconds at 98° C.

(3) 1 minute at 65° C.

(4) Repeating steps (2) and (3) nine times

(5) 5 minutes at 65° C.

The amplified products were purified using 18 μL of AMPure XP beads andeluting with 20 μL of a buffer comprising 10 mM Tris pH 8 and 0.05%Tween20.

The amplified products were assessed using a High Sensitivity DNA chipon 2100 Bioanalyzer (Agilent Genomics) and KAPA Library QuantificationKit for Illumina Platforms (Kapa Biosystems). The amplified productswere also diluted to 15 pM for sequencing on a MiSeq (Illumina)according to manufacturer's instructions (MiSeq Reagent Kit v3 2×75 bp)with a custom spike-in primer comprising the nucleotide sequence

(SEQ ID NO: 25) ACACTCTTTAAGACGACGTCGCTATGGCCTCTCC.

Example 3—Templated-Primer Extension Method for 96 Multiplexed Samples

In this example, a templated-primer extension method of the presentdisclosure was used to sequence identifier oligonucleotides collectedfrom 96 multiplexed samples. The single stranded nucleic acid templatesused in this example comprised a 3′ biotin moiety, a regioncomplementary to the unique nucleic acid sequences present in thecollected identifier oligonucleotides, a nucleic acid sequencecomprising a unique molecular identifier and a second amplificationprimer binding sequence (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, SEQ ID NO:26). Table 5 provides the sequences of the single-stranded nucleic acidtemplates used in this example.

TABLE 5 Single stranded nucleic acid templatesfor templated-primer extension method Single-stranded nucleic SEQ IDacid templates NO GTGACTGGAGTTCAGACGTGTGCTCTTCCG 27ATCTNNNNNNNNNNNNNNNTTGAAGCACAC CGTTTTTCTTTCTTCTTTCACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCG 28 ATCTNNNNNNNNNNNNNNNACCCACAGGTTATACGGGATTATCCGGTTATCCA GTGACTGGAGTTCAGACGTGTGCTCTTCCG 29ATCTNNNNNNNNNNNNNNNCGACACCGAGT TCGACCGTTATGTTGGTAGGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCG 30 ATCTNNNNNNNNNNNNNNNCGGTGTGTAAGCGTAACGATGTTGGTGTCGCTCT GTGACTGGAGTTCAGACGTGTGCTCTTCCG 31ATCTNNNNNNNNNNNNNNNCAGACACTGCG ACAACTCACGATCATGACACAGAGTGACTGGAGTTCAGACGTGTGCTCTTCCG 32 ATCTNNNNNNNNNNNNNNNATATTCTGTACTCAGTGCCTATCCACCTAATAGG GTGACTGGAGTTCAGACGTGTGCTCTTCCG 33ATCTNNNNNNNNNNNNNNNTTCAGTTATAA TGTGTCCAGCAGAAGCAGGAATTGTGACTGGAGTTCAGACGTGTGCTCTTCCG 34 ATCTNNNNNNNNNNNNNNNGTCCnTGTTGGGCGGACCGTAATGAGGAATTTG GTGACTGGAGTTCAGACGTGTGCTCTTCCG 35ATCTNNNNNNNNNNNNNNNGATGAGACTTC TACATGTCCGATGTTTTTGTGCTGTGACTGGAGTTCAGACGTGTGCTCTTCCG 36 ATCTNNNNNNNNNNNNNNNACTCACACATAGTACTGACACGTAAGATAGGATG GTGACTGGAGTTCAGACGTGTGCTCTTCCG 37ATCTNNNNNNNNNNNNNNNTTACCCTATCT CGTCTATGTACGTCAGGCTGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCG 38 ATCTNNNNNNNNNNNNNNNATCAACGTAGGGTAAGGTCATATTTTTACCTTAC GTGACTGGAGTTCAGACGTGTGCTCTTCCG 39ATCTNNNNNNNNNNNNNNNTTCCCTCTTTC TCCGCTTATGGATGAAAGGACAGGTGACTGGAGTTCAGACGTGTGCTCTTCCG 40 ATCTNNNNNNNNNNNNNNNCCTGCACAGTGAGTTTCTTTCACTCTAACTCTCT GTGACTGGAGTTCAGACGTGTGCTCTTCCG 41ATCTNNNNNNNNNNNNNNNTGTCGCTCTAG TGTGACTTTTCCACCTCGCATCTGTGACTGGAGTTCAGACGTGTGCTCTTCCG 42 ATCTNNNNNNNNNNNNNNNATATCTTTCTCGGGTAAAGATTAGGCGTCCGATA GTGACTGGAGTTCAGACGTGTGCTCTTCCG 43ATCTNNNNNNNNNNNNNNNCGATTAGCCGT AGACGCAACTCATTGCCGAAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCG 44 ATCTNNNNNNNNNNNNNNNTGTGAGCATTTCAGTACGAGTGATGCAGATAAAC GTGACTGGAGTTCAGACGTGTGCTCTTCCG 45ATCTNNNNNNNNNNNNNNNTATAGTTACCA AGTACTATGGGTTGGTGGAAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCG 46 ATCTNNNNNNNNNNNNNNNCCAATTATACTGTCTGTTATGTTCTCGGATAAGC GTGACTGGAGTTCAGACGTGTGCTCTTCCG 47ATCTNNNNNNNNNNNNNNNTCAGGTGCTTG TAGGCTCATGATAGGGGTAATGCGTGACTGGAGTTCAGACGTGTGCTCTTCCG 48 ATCTNNNNNNNNNNNNNNNCTCTGCTGTAATCTCAGCTCCACTTGTTTCTAAG GTGACTGGAGTTCAGACGTGTGCTCTTCCG 49ATCTNNNNNNNNNNNNNNNGTGCATATTGC AGCTGAGCCAGCTCAATTTGAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCG 50 ATCTNNNNNNNNNNNNNNNCCGTTGATTTACGCAACAGCGGCTTATATAGCTC GTGACTGGAGTTCAGACGTGTGCTCTTCCG 51ATCTNNNNNNNNNNNNNNNCATCATCGACA GTTCGCAGCCCTATAACATGATAGTGACTGGAGTTCAGACGTGTGCTCTTCCG 52 ATCTNNNNNNNNNNNNNNNATCGCAGGATGGTACAGCATCATACATGATGAGC GTGACTGGAGTTCAGACGTGTGCTCTTCCG 53ATCTNNNNNNNNNNNNNNNCTGATAAGTCG TAGGAATGTCGCTTAATACGGATGTGACTGGAGTTCAGACGTGTGCTCTTCCG 54 ATCTNNNNNNNNNNNNNNNATGGCGGTTTCGGGTCCTGCACTATTCCTAATAA GTGACTGGAGTTCAGACGTGTGCTCTTCCG 55ATCTNNNNNNNNNNNNNNNCCAGTACGGGT ACTAATAAGTGTCATATCTATTGGTGACTGGAGTTCAGACGTGTGCTCTTCCG 56 ATCTNNNNNNNNNNNNNNNTGTTGGAGAGGTTAGAGGTGAGGAGGCGAAGATA

Single stranded nucleic acid templates were ordered from Integrated DNATechnologies, Inc. and quantified using a NanoDrop 1000spectrophotometer (Thermo Fisher Scientific). Individual single strandednucleic acid templates were normalized to a standard concentration andthen pooled to be equimolar. The pool of single stranded nucleic acidtemplates was diluted to 0.83 nM in a buffer comprising 10 mM Tris pH 8and 0.05% Tween20.

The collected identifier oligonucleotides were hybridized to the singlestranded nucleic acid templates and extended by addition of 10 μL ofNEBNext Ultra II Q5 Master Mix (New England Biolabs), 4 μL of thediluted single stranded nucleic acid template pool and 4 μL of H₂O to 2μL of each sample of identifier oligonucleotides. The following PCRprogram was used to extend the identifier oligonucleotides:

(1) 30 seconds at 98° C., 10×

(2) 1 minutes at 98° C.,

(3) 1 minutes at 68° C.

(4) 1 minutes at 72° C.

(5) Repeating steps (2)-(4) ten times

(6) 2 minutes at 72° C.

The extension products were stored at 4° C. Magnetic streptavidin beads(MyOne Streptavidin Cl beads, Thermo Fisher Scientific) were washed in1× Binding and Washing Buffer (5 mM Tris-HCl, 0.5 mM EDTA, 1M NaCl), and5 μL of streptavidin beads were added to each extension product sample.The extension product samples were incubated with the beads on anorbital mixer for a minimum of 15 minutes. Following incubation, thesamples were heated to 95° C. for 3 minutes and transferred to amagnetic plate. Supernatant was extracted immediately after sufficientbead pelleting to yield the purified extension product samples.

The purified extension product samples were amplified by adding to 7.5μL of each purified extension product sample, 12.5 μL of NEBNext UltraII Q5 Master Mix, 0.25 μL of 100 forward primer, 1 μL of 25 μM reverseprimer and 3.8 μL of PCR-grade H₂O. The forward primer comprised a flowcell adapter sequence suitable for sequencing, a unique molecularidentifier and a nucleic acid sequence complementary to the firstamplification primer binding site located on the identifieroligonucleotide. Table 6 provides the sequences of the forward primersused in this example.

TABLE 6 Forward primers for templated- primer extension method SEQ IDPrimer Sequence NO AATGATACGGCGACCACCGAGATCTACACG 5CTCAGATATAGCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 6 CTCAGAATAGAGGCACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 7CTCAGACCTATCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 8 CTCAGAGGCTCTGAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 9CTCAGAAGGCGAAGACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 10 CTCAGATAATCTTAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 11CTCAGACAGGACGTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 12 CTCAGAGTACTGACACACTCTTTAAGACGACGTCGCTATGGCCTCTCC

The reverse primers comprised a flow cell adapter sequence suitable forsequencing, a unique molecular identifier and a nucleic acid sequencecomplementary to the second amplification primer binding site locatedsingle-stranded nucleic acid template. Table 7 provides the sequences ofthe reverse primers used.

TABLE 7 Reverse primers for templated- primer extension method SEQ IDPrimer Sequence NO CAAGCAGAAGACGGCATACGAGATGTCGGT 57AAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGGTCA 58CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGAATCC 59GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTACCT 60TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCATGAG 61GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGACTG 62ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCGTATT 63CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTCCTA 64GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTAGTTG 65CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGAGATA 66CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGGTGT 67ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTAATGC 68CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCAGAC 69GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGATAGG 70CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGGTAC 71AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCAAGGT 72CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCTATC 73CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATATGGAA 74GGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCAAGG 75ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTTACG 76CAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGTCTG 77TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCACGT 78AAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAACCTT 79GGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATATTGCG 80TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATACCTGG 81AAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGAGAT 82GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTACTC 83TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTAACG 84ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATATTCCT 85CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTGTTC 86CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAAGCAC 87TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTAGCA 88AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGCTTC 89CAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCTTAG 90CTGTGACTGGAGTTCAGACGTGTGctcttC CGATCT CAAGCAGAAGACGGCATACGAGATAACCGT 91TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGACATT 92CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGACCG 93TAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGATACT 94GGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGCGTA 95GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCGGTT 96ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATATGACG 97TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCTGTA 98AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCAATG 99GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATATCTCG 100CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGCTAT 101TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGTGTC 102TTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCAACT 103GGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTTCAC 104CAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATACGGTC 105TTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCTCGC 106AAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGAATT 107GCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATACGGAT 108TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTTAAGC 109GGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGCAGG 110TAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCAATCG 111ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTGCCA 112TAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGTTCG 113AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGGAGT 114TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATACGATG 115ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGATGT 116CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGAACC 117TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTTCGT 118TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTTCTG 119AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGCTCA 120TGGTGACTGGAGTTCAGACGTGTGctcttC CGATCT CAAGCAGAAGACGGCATACGAGATAGTTCG 121TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTAGCGT 122CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGCGTT 123ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGTGAT 124TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAACTTG 125CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCAAGA 126TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCGCAT 127TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGTACA 128CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGCTCC 129TAGTGAC'TGGAGTTCAGACGTGTGCTCTT CCGATCT CAAGCAGAAGACGGCATACGAGATGCAATT130 CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTTAGG131 ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTCCTA132 AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAACGTG133 GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTGTGT134 TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTTAAG135 GCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCACCTT136 ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGGTAG137 CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCAGTGA138 AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTTCAA139 CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGGCTA140 TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTGGAG141 TAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCTCTT142 CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCTAAC143 GCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGTCAG144 ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTCTGG145 TTGTGACTGGAGTTCAGACGTGTGctcttC CGATCT CAAGCAGAAGACGGCATACGAGATTGTGGT146 ACGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCCTATA147 CCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTTCTCT148 CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGTATGC149 TGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAAGTCG150 AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAACCGA151 AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTGTTGT152 GGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT

The PCR program used to amplify the purified extension productscomprised the following steps:

(1) 30 seconds at 98° C.

(2) 10 seconds at 98° C.

(3) 30 seconds at 65° C.

(4) 30 seconds at 72° C.

(5) Repeat steps (2)-(4) eighteen times

(6) 2 minutes at 72° C.

The amplified extension products were stored at 4° C. 4 μL of each PCRreaction were combined into four pools, for 24 samples per pool.

The pooled PCR reactions were purified using diluted Agencourt AMPure XPmagnetic beads (Beckman Coulter Genomics Inc.), which were prepared bycombining 100 μL of AMPure XP beads and 400 μL of AMPure XP buffer (2.5MNaCl, 20% PEG8000). Purification was performed with 76.8 μL of dilutedAMPure XP beads and eluted in 20 μL of a buffer comprising 10 mM Tris pH8 and 0.05% Tween20. Beads were retained and cleanup process wasrepeated with 24 μL of AMPure XP buffer and eluted in 20 μL of buffercomprising 10 mM Tris pH 8 and 0.05% Tween20.

The purified PCR products were assessed using a High Sensitivity DNAchip on 2100 Bioanalyzer (Agilent Genomics) and KAPA LibraryQuantification Kit for Illumina Platforms (Kapa Biosystems). Thepurified PCR products were also diluted to 15 pM for sequencing on aMiSeq (Illumina) according to manufacturer's instructions (MiSeq ReagentKit v3 2×75 bp) with a custom spike-in primer comprising the nucleotidesequence ACACTCTTTAAGACGACGTCGCTATGGCCTCTCC (SEQ ID NO: 25).

Example 4—Long Probe Hybridization Method for 96 Multiplexed Samples

In this example, a long probe hybridization method of the presentdisclosure was used to sequence identifier oligonucleotides collectedfrom 96 multiplexed samples. In this example, the first nucleic acidprobe comprises a 5′ phosphate moiety, a nucleic acid sequencecomplementary to a portion of the identifier oligonucleotide, a firstamplification primer binding site comprising an i7 sequence (SEQ ID NO:1), unique nucleic acid sequence which identifies the specific locationof the sample from which the identifier oligonucleotide was released anda P7 flow cell adapter sequence (SEQ ID NO: 4). The second nucleic acidprobe comprises a nucleic acid sequence complementary to a portion ofthe identifier oligonucleotide, a nucleic acid sequence comprising aunique molecular identifier, a second amplification primer binding sitecomprising and i5 sequence (SEQ ID NO: 2) and a P5 flow cell adaptersequence (SEQ ID NO: 3).

The first and second nucleic acid probes were ordered from IntegratedDNA Technologies, Inc. and quantified using a NanoDrop 1000spectrophotometer (Thermo Fisher Scientific). Individual nucleic acidprobes were normalized to a standard concentration, pooled to beequimolar, and diluted to 0.83 nM in a buffer comprising 10 mM Tris pH 8and 0.05% Tween20. The nucleic acid probes and the identifieroligonucleotides were hybridized by combining 0.5 μL of diluted nucleicacid probe pool with 2 μL of a mixture of identifier oligonucleotidescollected from a sample solution in a buffer comprising 50 mM NaCl. Thismixture was heated for 2 minutes at 95° C. and cooled for 30 minutes atambient temperature to yield an annealed identifieroligonucleotide-nucleic acid probe mixture.

In the case in which the first and the second nucleic acid probeshybridized to the identifier oligonucleotide such that the first and thesecond nucleic acid probes were not adjacent and were not overlapping, agap extension reaction was performed. To 2.5 μL of each annealedidentifier oligonucleotide-nucleic acid probe mixture, 3.8 μL of NEBNextUltra II Q5 Master mix and 1.3 μL of PCR-grade H₂O was added. Themixture was then subjected to the following Gap extension temperaturecycle:

(1) 30 seconds at 98° C.

(2) 1 minute at 98° C.

(3) 1 minute at 68° C.

(4) 1 minute at 72° C.,

(5) Repeat steps (2)-(4) ten times

(6) 2 minutes at 72° C.

The gap extension products were then stored at 4° C. The first andsecond nucleic acid probes were then ligated together by adding to 1 μLof the gap extension product, 10 μL of 2× rapid ligation buffer(Enzymatics), 1 μL of T4 DNA Rapid Ligase (Enzymatics), and 8 μL ofPCR-grade H₂O. These ligation reactions were incubated for 15 minutes20° C., subsequently quenched with 1 μL 0.5M EDTA, and pooled into asingle 15 mL conical tube.

In the case in which the first and the second nucleic acid probeshybridized to the identifier oligonucleotide such that the first and thesecond nucleic acid probes were adjacent and were not overlapping, nickrepair ligation reaction was performed. To 2.5 μL of each annealedidentifier oligonucleotide-nucleic acid probe mixture, 10 μL of 2× rapidligation buffer (Enzymatics), 1 μL of T4 DNA Rapid Ligase (Enzymatics),and 1 μL of PCR-grade H₂O was added. These ligation reactions wereincubated for 15 minutes at 20° C., subsequently quenched with 1 μL of0.5M EDTA, and pooled into a single 15 mL conical tube.

The pools of quenched ligation reactions were then purified usingdiluted Agencourt AMPure XP magnetic beads (Beckman Coulter GenomicsInc.), which were prepared by combining 350 μL of AMPure XP beads and3.15 mL of AMPure XP buffer (2.5M NaCl, 20% PEG8000). The purificationwas performed with 3.5 mL of diluted AMPure XP beads and eluted in 200μL of a buffer comprising 10 mM Tris pH 8 and 0.05% Tween20. The AMPureXP bead cleanup was then repeated with 400 μL of AMPure XP beads andeluted in 20 μL of a buffer comprising 10 mM Tris pH 8 and 0.05%Tween20.

To amplify the purified ligation products, PCR reactions with purifiedligation products and primers were prepared. To 6 μL of purifiedligation product, 10 μL of NEBNext Ultra II Q5 Master Mix, 0.2 μL of 100μM forward primer (CAAGCAGAAGACGGCATACGA, SEQ ID NO: 153) and reverseprimer (AATGATACGGCGACCACCGA, SEQ ID NO: 154) and 3.6 of PCR-grade H₂Owas added. The PCR program used to amplify the purified extensionproducts comprised the following steps:

(1) 30 seconds at 98° C.

(2) 10 seconds at 98° C.

(3) 30 seconds at 65° C.

(4) 30 seconds at 72° C.

(5) Repeat steps (2)-(4) eighteen times

(6) 2 minutes at 72° C.

The amplified products were stored at 4° C. 4 μL of each PCR reactionwere combined into six pools, for 16 samples per pool. The amplifiedproducts were further purified using an AMPure XP bead cleanup with 64μL of AMPure XP beads and eluting with 20 μL of a buffer comprising 10mM Tris pH 8 and 0.05% Tween20.

The purified amplified products were assessed using a High SensitivityDNA chip on 2100 Bioanalyzer (Agilent Genomics) and KAPA LibraryQuantification Kit for Illumina Platforms (Kapa Biosystems). Thepurified amplified products were also diluted to 15 pM for sequencing ona MiSeq (Illumina) according to manufacturer's instructions (MiSeqReagent Kit v3 2×75 bp) with either standard sequencing primers or acustom spike-in Read1 primer (SEQ ID NO: 25).

Example 5—Short Probe Hybridization Method for 96 Multiplexed Samples

In this example, a short probe hybridization method of the presentdisclosure was used to sequence identifier oligonucleotides collectedfrom 96 multiplexed samples. In this example, the first nucleic acidprobe comprises a 5′ phosphate moiety, a nucleic acid sequencecomplementary to a portion of the identifier oligonucleotide, a firstamplification primer binding site comprising an i7 sequence (SEQ IDNO: 1) and a unique nucleic acid sequence which identifies the specificlocation of the sample from which the identifier oligonucleotide wasreleased. The second nucleic acid probe comprises a nucleic acidsequence complementary to a portion of the identifier oligonucleotide, anucleic acid sequence comprising a unique molecular identifier and asecond amplification primer binding site comprising an i5 sequence (SEQID NO: 2).

The first and second nucleic acid probes were ordered from IntegratedDNA Technologies, Inc. and quantified using a NanoDrop 1000spectrophotometer (Thermo Fisher Scientific). Individual nucleic acidprobes were normalized to a standard concentration, pooled to beequimolar, and diluted to 0.83 nM in a buffer comprising 10 mM Tris pH 8and 0.05% Tween20. The nucleic acid probes and the identifieroligonucleotides were hybridized by combining 0.5 μL of diluted nucleicacid probe pool with 2 μL of a mixture of identifier oligonucleotidescollected from a sample solution in a buffer comprising 50 mM NaCl. Thismixture was heated for 2 minutes at 95° C. and cooled for 30 minutes atambient temperature to yield an annealed identifieroligonucleotide-nucleic acid probe mixture.

In the case in which the first and the second nucleic acid probeshybridized to the identifier oligonucleotide such that the first and thesecond nucleic acid probes were not adjacent and were not overlapping, agap extension reaction was performed. To 2.5 μL of each annealedidentifier oligonucleotide-nucleic acid probe mixture, 3.8 μL of NEBNextUltra II Q5 Master mix and 1.3 μL of PCR-grade H₂O was added. Themixture was then subjected to the following Gap extension temperaturecycle:

-   -   (1) 30 seconds at 98° C.    -   (2) 1 minute at 98° C.    -   (3) 1 minute at 68° C.    -   (4) 1 minute at 72° C.,    -   (5) Repeat steps (2)-(4) ten times    -   (6) 2 minutes at 72° C.

The gap extension products were then stored at 4° C. The first andsecond nucleic acid probes were then ligated together by adding to 1 μLof the gap extension product, 10 μL of 2× rapid ligation buffer(Enzymatics), 1 μL of T4 DNA Rapid Ligase (Enzymatics), and 8 μL ofPCR-grade H₂O. These ligation reactions were incubated 15 min 20° C.,quenched with 1 μL 0.5M EDTA, and pooled into a single 15 mL conicaltube.

In the case in which the first and the second nucleic acid probeshybridized to the identifier oligonucleotide such that the first and thesecond nucleic acid probes were adjacent and were not overlapping, nickrepair ligation reaction was performed. To 2.5 μL of each annealedidentifier oligonucleotide-nucleic acid probe mixture, 10 μL of 2× rapidligation buffer (Enzymatics), 1 μL of T4 DNA Rapid Ligase (Enzymatics),and 1 μL of PCR-grade H₂O was added. These ligation reactions wereincubated for 15 minutes at 20° C., subsequently quenched with 1 μL of0.5M EDTA, and pooled into a single 15 mL conical tube.

The pools of quenched ligation reactions were then purified usingdiluted Agencourt AMPure XP magnetic beads (Beckman Coulter GenomicsInc.), which were prepared by combining 350 μL of AMPure XP beads and3.15 mL of AMPure XP buffer (2.5M NaCl, 20% PEG8000). The purificationwas performed with 3.5 mL of diluted AMPure XP beads and eluted in 200μL of a buffer comprising 10 mM Tris pH 8 and 0.05% Tween20. The AMPureXP bead cleanup was then repeated with 400 μL of AMPure XP beads andeluted in 20 μL of a buffer comprising 10 mM Tris pH 8 and 0.05%Tween20.

To amplify the purified ligation products, PCR reactions with purifiedligation products and primers were prepared. To 6 μL of purifiedligation product, 10 μL of NEBNext Ultra II Q5 Master Mix, 0.2 μL of 100μM forward primer and reverse primer and 3.6 μL of PCR-grade H₂O wasadded. The forward primers comprised a flow cell adapter sequencesuitable for sequencing, a unique molecular identifier and a nucleicacid sequence complementary to the first amplification primer bindingsite located on the first strand of the nucleic acid adapter. Table 8provides the sequences of the forward primers used.

TABLE 8 Forward primers for short probe hybridization method SEQ IDPrimer Sequence NO CAAGCAGAAGACGGCATACGAGATCGAGTA 13ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTCTCCG 14GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAATGAG 15CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGGAATC 16TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTTCTGA 17ATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATACGAAT 18TCGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATAGCTTC 19AGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCGCAT 20TAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCATAGC 21CGGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATTTCGCG 22GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATGCGCGA 23GAGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT CAAGCAGAAGACGGCATACGAGATCTATCG 24CTGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCT

The reverse primers comprised a flow cell adapter sequence suitable forsequencing, a unique molecular identifier and a nucleic acid sequencecomplementary to the second amplification primer binding site located onthe second strand of the nucleic acid adapter. Table 9 provides thesequences of the reverse primers used.

TABLE 9 Reverse primers for short probe hybridization method SEQ IDPrimer Sequence NO AATGATACGGCGACCACCGAGATCTACACT 155ATAGCCTACACTCTTTCCCTACACGACGCT CTTCCGATCT AATGATACGGCGACCACCGAGATCTACACA156 TAGAGGCACACTCTTTCCCTACACGACGCT CTTCCGATCTAATGATACGGCGACCACCGAGATCTACACC 157 CTATCCTACACTCTTTCCCTACACGACGCTCTTCCGATCT AATGATACGGCGACCACCGAGATCTACACG 158GCTCTGAACACTCTTTCCCTACACGACGCT CTTCCGATCT AATGATACGGCGACCACCGAGATCTACACA159 GGCGAAGACACTCTTTCCCTACACGACGCT CTTCCGATCTAATGATACGGCGACCACCGAGATCTACACT 160 AATCTTAACACTCTTTCCCTACACGACGCTCTTCCGATCT AATGATACGGCGACCACCGAGATCTACACC 161AGGACGTACACTCTTTCCCTACACGACGCT CTTCCGATCT AATGATACGGCGACCACCGAGATCTACACG162 TACTGACACACTCTTTCCCTACACGACGCT CTTCCGATCT

The PCR program used to amplify the purified extension productscomprised the following steps:

(1) 30 seconds at 98° C.

(2) 10 seconds at 98° C.

(3) 30 seconds at 65° C.

(4) 30 seconds at 72° C.

(5) Repeat steps (2)-(4) eighteen times

(6) 2 minutes at 72° C.

The amplified products were stored at 4° C. 4 μL of each PCR reactionwere combined into six pools, for 16 samples per pool. The amplifiedproducts were further purified using an AMPure XP bead cleanup with 64μL of AMPure XP beads and eluting with 20 μL of a buffer comprising 10mM Tris pH 8 and 0.05% Tween20.

The purified amplified products were assessed using a High SensitivityDNA chip on 2100 Bioanalyzer (Agilent Genomics) and KAPA LibraryQuantification Kit for Illumina Platforms (Kapa Biosystems). Thepurified amplified products were also diluted to 15 pM for sequencing ona MiSeq (Illumina) according to manufacturer's instructions (MiSeqReagent Kit v3 2×75 bp) with either standard sequencing primers or acustom spike-in Read1 primer (SEQ ID NO: 25).

Example 6—Direct PCR Method for 96 Multiplexed Samples

In this example, a direct PCR method of the present disclosure was usedto sequence identifier oligonucleotides collected from 96 multiplexedsamples. In this example, 8 species of forward amplification primers and12 species of reverse amplification primers were used. The forwardprimers comprised a P5 flow cell adapter (SEQ ID NO: 3), a nucleic acidsequence comprising a unique molecular identifier and a regioncomplementary to a first amplification primer binding site present onthe identifier oligonucleotide. Table 10 provides the sequences of theforward amplification primers used.

TABLE 10 Forward amplification primers for direct PCR method SEQ IDPrimer Sequence NO AATGATACGGCGACCACCGAGATCTACACG 5CTCAGATATAGCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 6 CTCAGAATAGAGGCACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 7CTCAGACCTATCCTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 8 CTCAGAGGCTCTGAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 9CTCAGAAGGCGAAGACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 10 CTCAGATAATCTTAACACTCTTTAAGACGACGTCGCTATGGCCTCTCC AATGATACGGCGACCACCGAGATCTACACG 11CTCAGACAGGACGTACACTCTTTAAGACGA CGTCGCTATGGCCTCTCCAATGATACGGCGACCACCGAGATCTACACG 12 CTCAGAGTACTGACACACTCTTTAAGACGACGTCGCTATGGCCTCTCC

The reverse primers comprised a P7 flow cell adapter (SEQ ID NO: 4), anucleic acid sequence comprising a unique molecular identifier and aregion complementary to a second amplification primer binding sitepresent on the identifier oligonucleotide. Table 11 provides thesequences of the reverse amplification primers used.

TABLE 11 Reverse amplification primers for direct PCR method SEQ IDPrimer Sequence NO CAAGCAGAAGACGGCATACGAGATCGTGAT 163GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAATCAAGCAGAAGACGGCATACGAGATACATCG 164 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAAT CAAGCAGAAGACGGCATACGAGATGCCTAA165 GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGATGCAGCAAAAT CAAGCAGAAGACGGCATACGAGATTGGTCA 166GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAATCAAGCAGAAGACGGCATACGAGATCACTGT 167 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAAT CAAGCAGAAGACGGCATACGAGATATTGGC168 GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGATGCAGCAAAAT CAAGCAGAAGACGGCATACGAGATGATCTG 169GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAATCAAGCAGAAGACGGCATACGAGATTCAAGT 170 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAAT CAAGCAGAAGACGGCATACGAGATCTGATC171 GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGATGCAGCAAAAT CAAGCAGAAGACGGCATACGAGATAAGCTA 172GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAATCAAGCAGAAGACGGCATACGAGATGTAGCC 173 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNNNAACGGACAGGAT GCAGCAAAAT CAAGCAGAAGACGGCATACGAGATTACAAG174 GTGACTGGAGTTCAGACGTGTGCTCTTCCG ATCTNNNNNNNNNNNNNNAACGGACAGGATGCAGCAAAAT

In the case of 8 forward amplification primers and 12 reverseamplification primers, when the unique molecular identifiers from a pairof forward and reverse primers are combined, a total of 96 uniquecombinations can be obtained, allowing for the multiplexing of 96samples.

To amplify the collected identifier oligonucleotides for sequencing, PCRreactions with collected identifier oligonucleotides and forward andreverse amplification primers were prepared on a 96-well plate with 2 μLof each identifier oligonucleotide sample, 10 μL of NEBNext Ultra II Q5Master Mix, 2 μL of 10 μM forward amplification primer, 2 μL of reverseamplification primer, and 4 μL of PCR-grade H₂O. Each well in the96-well plate contained an identifier oligonucleotide sample and aunique combination of forward and reverse amplification primers. The PCRprogram used comprised the following steps:

(1) 30 seconds at 98° C.

(2) 10 seconds at 98° C.

(3) 30 seconds at 65° C.

(4) 30 seconds at 72° C.

(5) Repeat steps (2)-(4) six to ten times

(6) 2 minutes at 72° C.

The amplified products were stored at 4° C. 10 μL of each PCR reactionwas combined into a single 15 mL conical tube.

The pooled PCR reactions were purified using diluted Agencourt AMPure XPmagnetic beads (Beckman Coulter Genomics Inc.), which were prepared bycombining 115.2 μL of AMPure XP beads and 1036.8 μL of AMPure XP buffer(2.5M NaCl, 20% PEG8000). Purification was performed with 1152 μL ofdiluted AMPure XP beads and eluted in 60 μL of a buffer comprising 10 mMTris pH 8. The purification process was repeated with 60 μL of AMPure XPbeads and eluted in 70 μL of a buffer comprising 10 mM Tris pH 8.

Following AMPure XP cleanup, PCR reactions with universal primers wereprepared with 9 μL of pooled direct PCR product, 15 μL of NEBNext UltraII Q5 Master Mix, 3 μL of 10 universal P7 primer (SEQ ID NO: 153) and 2μL of 10 μM universal P5 primer (SEQ ID NO: 154). The PCR program usedwas:

(1) 30 seconds at 98° C.,

(2) 10 seconds at 98° C.

(3) 30 seconds at 65° C.

(4) 30 seconds at 72° C.

(5) Repeat steps (2)-(4) 15 to 24 times

(6) 2 minutes at 72° C.

Two rounds of AMPure XP bead cleanup was performed. The first round wasperformed with 30 μL of beads and eluted with 20 μL of a buffercomprising 10 mM Tris pH 8 and the second round was performed with 20 μLbeads and eluted with 11 μL of a buffer comprising 10 mM Tris pH 8.

These purified PCR products were assessed using a High Sensitivity DNAchip on 2100 Bioanalyzer (Agilent Genomics). The purified PCR productswere also diluted for sequencing on a MiSeq (Illumina) according tomanufacturer's instructions (MiSeq Reagent Kit v3 2×75 bp) with a customspike-in primer (SEQ ID NO: 25).

Example 7—Spatially Detecting Target Analytes in a FFPE Sample

The methods of the present invention were used to spatially detect aplurality of different target analytes, including target proteins andtarget RNAs, in a sample of inflamed human tonsil tissue FFPE section.

In one experiment, 30 different target proteins were spatially detectedusing the methods of the present disclosure in two serial sections cutfrom the inflamed human tonsil tissue FFPE section. The 30 targetproteins are put forth in Table 12. The 30 target proteins included IgGRabbit isotype and IgG Mouse isotype as negative controls, as thesetarget proteins should not have been present in the inflamed humantonsil sample and therefore should not have been detected.

TABLE 12 Target Proteins Target Protein Target Protein AKT FOXP3 B7-H3GZMB Bcl-2 Histone H3 Beta-2-microglobulin Ki67 Beta-catenin CD20 CD14P-AKT CD19 PanCK CD3 PD1 CD4 PD-L1 CD44 S6 CD45 STAT3 CD45RO P-STAT3CD56 VISTA CD68 IgG Rabbit isotype (negative control) CD8A IgG Mouseisotype (control)

To spatially detect the 30 target proteins, 30 different probes of thepresent disclosure were used. Each probe comprised a target bindingdomain comprising an antibody that specifically binds to one of the 30target proteins in Table 12. The two serial sections were contacted witha plurality of the 30 different probes. Ninety-six regions of interest(ROI) were then identified. For each ROI, the ROI was illuminated withUV light to release the identifier oligonucleotides from the probesbound within the ROI. The released identifier oligonucleotides were thencollected and identified using a short probe hybridization method of thepresent disclosure, thereby spatially detecting the 30 target proteinsin the two serial sections. As shown in FIGS. 21A-21D, the number ofreads per target protein in each ROI for the two serial sections werewell correlated, demonstrating that the method yields reproducibleresults.

In a second experiment, 20 different target RNAs were spatially detectedusing the methods of the present disclosure in two different serialsections cut from the inflamed human tonsil tissue FFPE section. The 20different target RNAs are put forth in Table 13. The 20 target RNAsincluded 6 negative controls (Negative Probe) that should not have beendetected in the sample.

TABLE 13 Target RNAs Target RNA Target RNA Target RNA CD3E CD40 CTLA4CD3G CD45 GAPDH CD4 CD74 KRT13 CD20 CD79A PD1 PSA RP56 Negative Probe #1Negative Probe #2 Negative Probe #3 Negative Probe #4 Negative Probe #5Negative Probe #6

To spatially detect the 20 Target RNAs, 20 different probes of thepresent disclosure were used. Each probe comprised a target bindingdomain comprising a nucleic acid sequence complementary to at least oneportion of one of the 20 target RNAs. The two serial sections werecontacted with a plurality of the 20 different probes. Ninety-sixregions of interest (ROI) were then identified. For each ROI, the ROIwas illuminated with UV light to release the identifier oligonucleotidesfrom the probes bound within the ROI. The released identifieroligonucleotides were then collected and identified using a direct PCRmethod of the present disclosure, thereby spatially detecting the 20target RNAs in the two serial sections. As shown in FIGS. 22A-22D, thenumber of reads per target RNA in each ROI for the two serial sectionswere well correlated, demonstrating that the method yields reproducibleresults.

Example 8—Spatially Detecting Target Proteins in a Fluorescently StainedFFPE Sample

In another experiment, a 5 μm FFPE section of inflamed human tonsiltissue was stained with 4 fluorescent visualization markers: (1) CD3E, aT-cell marker; (2) PanCK, an epithelial cell marker; (3) Ki-67, aproliferation marker; and (4) SYTO83, a DNA stain, as shown in the leftpanel of FIG. 23. The stained FFPE section was then contacted with theprobes directed against 30 target proteins, as described in Example 7.As shown in the left panel FIG. 23, 96 regions of interest (ROIs) wereselected. Each ROI was a circle with a 500 μm diameter. For each ROI,the ROI was illuminated with UV light to release the identifieroligonucleotides from the probes bound within the ROI. The releasedidentifier oligonucleotides were then collected and identified using ashort probe hybridization method of the present disclosure, therebyspatially detecting the 30 target proteins in the FFPE section. As shownin the right panel of FIG. 23, PanCK, CD3E and Ki67 were spatiallydetected in ROIs that correlated with their fluorescent visualizationmarkers. Thus, the results generated by the methods of the presentdisclosure correlate with the results generated using establishedimmunohistochemical method.

Example 9—Spatially Detecting Target RNAs in a FFPE Sample

In another experiment, a 5 μm section from an inflamed human tonsiltissue FFPE block was contacted with probes directed against 20 targetRNAs, as described in Example 7. 96 regions of interest (ROIs) were thenselected. Each ROI was a circle with a 500 μm diameter. For each ROI,the ROI was illuminated with UV light to release the identifieroligonucleotides from the probes bound within the ROI. The releasedidentifier oligonucleotides were then collected and identified using adirect PCR method of the present disclosure, thereby spatially detectingthe 20 target RNAs in the two serial sections. The total RNA from a 20μm section from the same inflamed human tonsil tissue FFPE block wasthen isolated. The total RNA was analyzed using the NanoString nCounter®system. FIG. 24 shows that the average number of counts for 11 differentRNA targets recorded using the methods of the present disclosure werewell correlated with the average number of counts for the same 11different RNA targets recorded using the nCounter® system. Thus, theresults generated using the methods of the present disclosure correlatewith the results generated using established direct detection methods.

Example 10—Spatially Detecting Target RNAs in Specific Sub-Regions of anROI

In another experiment, a 5 μm section from an inflamed human tonsiltissue FFPE block was contacted with probes directed against 30 targetproteins, as described in Example 7. The same 5 μm section was alsostained with 4 fluorescent visualization markers: (1) CD3E, a T-cellmarker; (2) PanCK, an epithelial cell marker; (3) Ki-67, a proliferationmarker; and (4) SYTO83, a DNA stain. As shown in FIG. 25, 48 regions ofinterest (ROIs) were identified. For each ROI, two sub-regions were thenidentified based on the fluorescent staining. Areas of an ROI that werefluorescently stained positive for PanCK (PanCK+) were designated a“tumor” sub-region and the areas of an ROI that lacked PanCK fluorescentstaining were designated a “micro-environment” sub-region, as shown inFIG. 25. For each ROI, the tumor sub-region and the micro-environmentsub-region were separately illuminated with UV light to release theidentifier oligonucleotides from the probes bound within each sub-regionby creating a custom mask based on the intensity of PanCK fluorescentstaining. The released identifier oligonucleotides were also separatelycollected. The collected identifier oligonucleotides were then analyzedusing the short-probe hybridization method of the present disclosure andthe NanoString nCounter® system. As shown in the bottom panel of FIG.25, the results using the NanoString nCounter® system and theshort-probe hybridization method of the present disclosure were wellcorrelated. Furthermore, in the tumor sub-regions, PanCK was detected ata significantly higher level as compared to the micro-environmentsub-regions. Thus, the spatial detection results provided by the methodsof the present disclosure are consistent with established fluorescentimmunohistochemical methods and allows for the spatial detection withinhighly specific regions of a sample.

Example 11—96-Plex Human Immuno-Oncology Panel

A 96-plex human immuno-oncology panel was designed for use in thedirect-PCR methods of the present invention. The panel comprised aplurality of probes that could be used to spatially detect 96 differenthuman target RNAs using the direct-PCR methods of the presentdisclosure. The 96 target RNAs are shown in Table 14.

TABLE 14 Target RNAs Target AKT1 ARG1 B2M BATF3 BCL2 BCL2L1 CCL5 CCND1CD14 CD27 CD274 CD276 CD3E CD4 CD40 CD40LG CD44 CD47 CD68 CD74 CD86 CD8ACEACAM1 CEACAM6 CEACAM8 CMKLR1 CSF1R CTLA4 CTNNB1 CXCL10 CXCL9 CSCR6DKK2 EPCAM FAS FASLG FOXP3 GZMB H3F3A HAVCR2 HIF1A HLA_DQA1 HLA_DRB HLA_E ICAM1 ICOSLG IDO1 IFNAR1 IFNG IFNGR1 IL10 IL12B IL15 IL1B IL6 ITGAMITGAV ITGAX ITGB2 ITGB8 KRT1 KRT10 KRT14 KRT17 KRT18 KRT19 KRT6A KRT7LAG3 LY6E MKI67 MS4A1 NCAM1 NKG7 NT5E PDCD1 PDCD1LG2 PECAM1 PMEL PSMB10PTEN PRTPRC RPS6 S100B SOD2 SOX10 STAT1 STAT2 STAT3 TBX21 TIGIT TNFTNFRSF9 TNFSF4 VEGFA VSIR

In total, the panel comprised 928 different probes. Each of the probescomprised an identifier oligonucleotide comprising a first amplificationprimer binding site, a nucleic acid sequence comprising a uniquemolecular identifier, a unique nucleic acid sequence which identifiedthe target RNA bound to the target binding domain and a secondamplification primer binding site. FIG. 26 shows a schematic of theprobes used in the panel. For each of the 96 target RNAs, there was atleast one probe within the 928 probe set comprising a target bindingdomain that directly or indirectly hybridized to that target RNA. Formost of the 96 target RNAs, there were 10 different probes that directlyor indirectly hybridized to the specific target RNA. These 10 differentprobes directly or indirectly hybridized to different locations on thetarget RNA to create a “tiling” effect, as shown in the top panel ofFIG. 27. Tiling multiple probes onto a target RNA means that each targetRNA will be individually detected multiple times, increasing the overallaccuracy of the measurement. For example, as shown in the bottom panelof FIG. 27, in the case where 10 probes are tiled onto a single targetRNA, one of the probes may be incorrectly detected too many times(outlier high count probe), while another probe may be incorrectlydetected too few times (outlier low count probe). However, the other 8probes may be detected at a similar level, indicating that the twooutliers should be discarded during analysis and the signals from the 8probes used to generate a more accurate measurement of the abundance ofthe target RNA.

The set of 928 probes also comprised 80 negative control probes. Each ofthe 80 negative control probes comprised a target binding domaincomprising a scrambled, non-specific nucleic acid sequence that wasdesigned using guidelines from the External RNA Controls Consortium suchthat the target binding domain should not be complementary to RNAmolecules present within a human sample. Thus, these 80 negative controlprobes should not be detected during analysis.

The 96-plex human immune-oncology panel was used to analyze a tissuemicroarray. The tissue microarray comprised FFPE samples of 22 commonhuman cell lines, including normal and cancerous cell types. Some of thecell lines are shown in Table 15.

TABLE 15 Cell lines Cell Line Cell Line Cell Line Cell Line CCRF-CEMDAUDI H596 H2228 HT29 HUT78 HUH7 JURKAT M14 MDA-MB-468 MOLT4 RAJI SKBR3SUDHL1 SUDHL4

The tissue microarray also comprised one mouse cell line (3T3) as anegative control. Each of the FFPE samples on the microarray wascontacted with a plurality of the 928 different probes in theimunno-onocology panel. As shown in FIG. 28, for each of the FFPEsamples, at least three circular regions of interest (ROIs) with adiameter of 300 μm were selected. As a negative control, ROIs were alsoselected on regions of the microarray that did not comprise a FFPEsample (glass negative control). For each ROI, the ROI was illuminatedwith UV light to release the identifier oligonucleotides from the probesbound within the ROI. The released identifier oligonucleotides were thencollected and identified using a direct PCR method of the presentdisclosure thereby spatially detecting the 96 target RNAs in each of theFFPE samples on the tissue microarray.

FIG. 29 shows that a sufficient read depth was achieved using a MiSeq v3flowcell. The top panel of FIG. 30 shows that none of the target RNAswere spatially detected for the glass negative control ROIs. Likewise,the bottom panel of FIG. 30 shows that nearly none of the target RNAswere detected in the negative control mouse 3T3 FFPE sample. Conversely,as shown in FIGS. 31, 33 and 34, specific target RNAs were successfullydetected in the HEK293 (human embryonic kidney) FFPE sample and theJurkat (human T-cell lymphocyte) FFPE sample. FIGS. 31, 33 and 34 showthat clusters of “tiled” probes were detected for particular targetRNAs, including AKT1, B2M, CD3E, HIF1A, PTEN, RPS6, STAT1, STAT2, STAT3,VEGF, PTPRC (CD45), and KRT1/10/18/19. These results indicated thatthere are certain target RNAs that are differentially transcribed in thetwo different cells lines. The results of this experiment were alsoverified using the NanoString nCounter system to identify the collectedidentifier oligonucleotides. As shown in FIG. 27, the results using thedirect-PCR method of the present disclosure were consistent with theresults obtained using the NanoString nCounter system.

What is claimed is:
 1. A method comprising: a) collecting a plurality ofoligonucleotides from a first location of a tissue sample underconditions that release the plurality of oligonucleotides from the firstlocation of the tissue sample; b) collecting a plurality ofoligonucleotides from a second location of the tissue sample underconditions that release the plurality of oligonucleotides from thesecond location of the tissue sample; c) synthesizing a first pluralityof DNA products by performing a synthesis reaction that uses theplurality of oligonucleotides collected in step (a) as templates andincorporates at least one nucleic acid sequence that identifies thefirst location of the tissue sample into each of the first plurality ofDNA products; d) synthesizing a second plurality of DNA products byperforming a synthesis reaction that uses the plurality ofoligonucleotides collected in step (b) as templates and incorporates atleast one nucleic acid sequence that identifies the second location ofthe tissue sample into each of the second plurality of DNA products; ande) identifying the first plurality of DNA products and the secondplurality of DNA products synthesized in step (c) and step (d) bysequencing the first plurality of DNA products and the second pluralityof DNA products, thereby spatially detecting the plurality ofoligonucleotides collected from the first location of the tissue sampleand the plurality of oligonucleotides collected from the second locationof the tissue sample.
 2. The method of claim 1, wherein the tissuesample is immobilized onto a microscope slide.
 3. The method of claim 2,wherein the microscope slide comprises a plurality of primersimmobilized on the microscope slide.
 4. The method of claim 3, whereinthe plurality of primers is immobilized on the microscope slide at their5′ ends.
 5. The method of claim 4, wherein each of step (c) and step (d)comprises performing a solid-phase amplification reaction, wherein thesolid-phase amplification reaction is carried out on the microscopeslide using the plurality of primers immobilized on the microscopeslide.
 6. The method of claim 1, wherein the sequencing step isperformed using a next generation sequencing reaction.
 7. The method ofclaim 1, further comprises amplifying a library using the firstplurality of DNA products and the second plurality of DNA products astemplates.
 8. The method of claim 1, wherein the at least one nucleicacid sequence that identifies the first location of the tissue samplecomprise at least one unique molecular identifier.
 9. The method ofclaim 1, wherein the at least one nucleic acid sequence that identifiesthe second location of the tissue sample comprise at least one uniquemolecular identifier.
 10. The method of claim 1, wherein the firstplurality of DNA products further comprises at least one amplificationprimer binding site.
 11. The method of claim 1, wherein the secondplurality of DNA products further comprises at least one amplificationprimer binding site.
 12. The method of claim 1, wherein steps (a) and(b) are performed simultaneously.
 13. The method of claim 1, whereinsteps (c) and (d) are performed simultaneously.